Human aminoacyl-tRNA synthetase polypeptides useful for the regulation of angiogenesis

ABSTRACT

The invention provides compositions comprising truncated tryptophanyl tRNA synthetase polypeptides useful for regulating angiogenesis, as well as nucleic acids encoding such truncated tRNA synthetase polypeptides. Methods of making and using such compositions are also disclosed.

GOVERNMENTAL RIGHTS

[0001] This invention was made with governmental support from the UnitedStates Government, National Institutes of Health, Grant GM23562; theUnited States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to compositions comprising truncated tRNAsynthetase polypeptides, as well as nucleic acids encoding suchtruncated tRNA synthetase polypeptides. Methods of making and using suchcompositions are also disclosed.

[0004] 2. Background of the Invention

[0005] Aminoacyl-tRNA synthetases, which catalyze the aminoacylation oftRNA molecules, are ancient proteins that are essential for decodinggenetic information during the process of translation. Inhighereukaryotes, nine aminoacyl-tRNA synthetases associate with atleastthree other polypeptides to form a supramolecular multienzyme complex(Mirande et al., 1985, Eur. J. Biochem. 147:281-89). Each of theeukaryotic tRNA synthetases consists of a core enzyme, which is closelyrelated to the prokaryotic counterpart of the tRNA synthetase, and anadditional domain that is appended to the amino-terminal orcarboxyl-terminal end of the core enzyme (Mirande, 1991, Prog. NucleicAcid Res. Mol. Biol. 40:95-142). Human tyrosyl-tRNA synthetase (TyrRS),for example, has a carboxyl-terminal domain that is not part ofprokaryotic and lower eukaryotic TyrRS molecules (FIG. 1) (Rho et al.,1998, J. Biol. Chem. 273:11267-73). It has also been suggested that boththe bovine and rabbit TyrRS molecules possess an extra domain (Kleemanet al., 1997, J. Biol. Chem. 272:14420-25).

[0006] In most cases, the appended domains appear to contribute to theassembly of the multienzyme complex (Mirande, supra). However, thepresence of an extra domain is not strictly correlated with theassociation of a synthetase into the multienzyme complex. Highereukaryotic TyrRS, for example, is not a component of the multienzymecomplex (Mirande et al., supra).

[0007] The carboxyl-terminal domain of human TyrRS shares a 51% sequenceidentity with the mature form of human endothelial monocyte-activatingpolypeptide II (EMAP II) (Rho et al., supra). TyrRS is the only highereukaryotic aminoacyl-tRNA synthetase known to contain an EMAPII-likedomain. The EMAP-like domain of TyrRS has been shown to be dispensablefor aminoacylation in vitro and in yeast (Wakasugi et al., 1998, EMBO J.17:297-305).

[0008] EMAP II is a proinflammatory cytokine that was initiallyidentified as a product of murine methylcholanthrene A-inducedfibrosarcomacells. Pro-EMAP II is cleaved and is secreted from apoptoticcells to produce a biologically active 22-kD mature cytokine (Kao, etal., 1994, J. Biol. Chem. 269:25106-19). The mature EMAP II can inducemigration of mononuclear phagocytes (MPs) andpolymorphonuclearleukocytes (PMNs); it also stimulates the production oftumor necrosis factor-α (TNFα) and tissue factor by MPs and the releaseof myeloperoxidase from PMNs.

[0009] The catalytic core domain of tryptophanyl-tRNA synthetase (TrpRS)is a close homolog of the catalytic domain of TyrRS (Brown et al., 1997,J. Mol. Evol. 45:9-12). As shown in FIG. 15, mammalian TrpRS moleculeshave an amino-terminal appended domain. In normal human cells, two formsof TrpRS can be detected: a major form consisting of the full-lengthmolecule and a minor truncated form (“mini TrpRS”). The minor form isgenerated by the deletion of the amino-terminal domain throughalternative splicing of the pre-mRNA (Tolstrup et al., 1995, J. Biol.Chem. 270:397-403). The amino-terminus of mini TrpRS has been determinedto be the met residue at position 48 of the full-length TrpRS molecule(id.). Alternatively, truncated TrpRS may be generated by proteolysis(Lemaire et al., 1975, Eur. J. Biochem. 51:237-52). For example, bovineTrpRS is highly expressed in the pancreas and is secreted into thepancreatic juice (Kisselev, 1993, Biochimie 75:1027-39), thus resultingin the production of a truncated TrpRS molecule. These results suggestthat truncated TrpRS has a function other than the arninoacylation oftRNA (Kisselev, supra).

[0010] Angiogenesis, or the proliferation of new capillaries frompre-existing blood vessels, is a fundamental process necessary forembryonic development, subsequent growth, and tissue repair.Angiogenesis is a prerequisite for the development and differentiationof the vascular tree, as well as for a wide variety of fundamentalphysiological processes including embryogenesis, somatic growth, tissueand organ repair and regeneration, cyclical growth of the corpus luteumand endometrium, and development and differentiation of the nervoussystem. In the female reproductive system, angiogenesis occurs in thefollicle during its development, in the corpus luteum followingovulation and in the placenta to establish and maintain pregnancy.Angiogenesis additionally occurs as part of the body's repair processes,e.g. in the healing of wounds and fractures. Angiogenesis is also afactor in tumor growth, since a tumor must continuously stimulate growthof new capillary blood vessels in order to grow. Angiogenesis is anessential part of the growth of human solid cancer, and abnormalangiogenesis is associated with other diseases such as rheumatoidarthritis, psoriasis, and diabetic retinopathy (Folkman, J. andKlagsbrun, M., Science 235:442-447,(1987)).

[0011] Several factors are involved in angiogenesis. Both acidic andbasic fibroblast growth factor molecules that are mitogens forendothelial cells and other cell types. Angiotropin and angiogenin caninduce angiogenesis, although their functions are unclear (Folkman, J.,1993, Cancer Medicine pp. 153-170, Lea and Febiger Press). A highlyselective mitogen for vascular endothelial cells is vascular endothelialgrowth factor or VEGF (Ferrara, N., et al., Endocr. Rev. 13:19-32,(1992)).

SUMMARY OF THE INVENTION

[0012] The invention provides novel truncated tRNA synthetasepolypeptides having chemokine activity that are useful for research,diagnostic, prognostic and therapeutic applications. In one embodiment,the tRNA synthetase polypeptides are useful for regulating vascularendothelial cell function, and in particular, for regulatingangiogenesis.

[0013] The truncated tryptophanol tRNA synthetase derived polypeptideshave an amino-terminal truncation but may include a Rossmann foldnucleotide binding domain. The isolated polypeptide is capable ofregulating vascular endothelial cell function and preferably has a sizeof at least bout 46 kiloDaltons (kD).

[0014] Preferred truncated tryptophanyl tRNA synthetase polypeptidesinclude a polypeptide consisting essentially of amino acid residues48-471 of SEQ ID NO:9; a polypeptide consisting essentially of aminoacid residues 71-471 of SEQ ID NO:9; a polypeptide of approximately 47kD molecular weight produced by cleavage of the polypeptide of SEQ IDNO:9 with polymorphonuclear leucocyte elastase; and fragments thereofcomprising the amino acid sequence -Asp-Leu-Thr-. In one preferredembodiment, the truncated tRNA synthetase polypeptide is mammalian, andmore preferably, human.

[0015] In another embodiment, the invention comprises an isolatednucleic acid molecule comprising a polynucleotide having a nucleotidesequence at least 95% identical to a sequence selected from the groupconsisting of a polynucleotide of SEQ ID NO:9, a polynucleotide which ishybridizable to a polynucleotide of SEQ ID NO:9, a polynucleotideencoding a truncated tryptophanyl-tRNA synthetase polypeptide whichincludes a Rossmann fold nucleotide binding domain, a polynucleotidethat is hybridizable to a polynucleotide encoding a truncatedtryptophanyl-tRNA synthetase polypeptide which includes a Rossmann foldnucleotide binding domain, a polynucleotide encoding a polypeptidementioned in the preceding paragraph, a polynucleotide that ishybridizable to a polynucleotide encoding a polypeptide mentioned in thepreceding paragraph, a polynucleotide encoding a polypeptide epitope ofSEQ ID NO:9, and a polynucleotide that is hybridizable to apolynucleotide encoding a polypeptide epitope of SEQ ID NO:9. In apreferred embodiment the invention comprises a recombinant expressionvector comprising the isolated nucleic acid molecule of encoding a tRNAsynthetase polypeptide. Another embodiment is a host cell comprising arecombinant expression vector comprising the isolated nucleic acidmolecule of SEQ ID NO:9 encoding a tRNA synthetase polypeptide.

[0016] In one embodiment, the present invention is a process for makingtRNA synthetase polypeptides by treating tryptophanyl-tRNA synthetasewith a protease. One preferred protease is polymorphonuclear leukocyteelastase.

[0017] The invention provides compositions comprising tryptophanyl-tRNAsynthetase polypeptides and a pharmaceutically suitable excipient. Suchcompositions are suitable for transdermal, transmucosal, enteral orparenteral administration. In another embodiment, the tRNA synthetasepolypeptide can be used for the preparation of a pharmaceuticalcomposition for transdermal, transmucosal, enteral or parenteraladministration.

[0018] In one embodiment, the tRNA synthetase polypeptide can haveangiogenic activity at least two-fold greater than control levels. Inembodiments in which the tRNA synthetase polypeptide has angiostaticactivity, the polypeptide suppresses at least ten percent of angiogenicactivity, more preferably at least ninety percent of angiogenicactivity.

[0019] The invention further provides a method of enhancing angiogenesisin a mammal comprising the step of administering an angiogenicallyeffective amount of a composition comprising an angiogenic tRNAsynthetase polypeptide and a pharmaceutically suitable excipient.

[0020] The invention further provides a method of suppressingangiogenesis in a mammal comprising the step of administering anangiostatically effective amount of a composition comprising anangiostatic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

[0021] In another embodiment, the invention provides a method ofenhancing angiogenesis to a graft in a mammal comprising the step ofadministering an angiogenically effective amount of a compositioncomprising an angiogenic tRNA synthetase polypeptide and apharmaceutically suitable excipient.

[0022] In another embodiment, the invention provides a method oftreating myocardial infarction in a mammal comprising the step ofadministering an angiogenically effective amount of a compositioncomprising an angiogenic tRNA synthetase polypeptide and apharmaceutically suitable excipient.

[0023] In another embodiment, the invention provides a method oftreating a condition that would benefit from increased angiogenesis in amammal comprising the step of administering an angiogenically effectiveamount of a composition comprising an angiogenic TRNA synthetasepolypeptide and a pharmaceutically suitable excipient.

[0024] In another embodiment, the invention provides a method oftreating a condition that would benefit from decreased angiogenesis in amammal comprising the step of administering an angiostatically effectiveamount of the composition comprising an angiostatic tRNA synthetasepolypeptide and a pharmaceutically suitable excipient.

[0025] In another embodiment, the invention provides a method oftreating a solid tumor in a mammal comprising the step of administeringan angiostatically effective amount of the composition comprising anangiostatic tRNA synthetase polypeptide and a pharmaceutically suitableexcipient.

[0026] In another embodiment, the invention provides a method ofsuppressing tumor metatasis in a mammal comprising the step ofadministering an angiostatically effective amount of the compositioncomprising an angiostatic tRNA synthetase polypeptide and apharmaceutically suitable excipient.

DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 illustrates a schematic alignment of E. coli TyrRS, humanfull-length TyrRS, human mini TyrRS, the human TyrRS carboxyl-terminaldomain, and human mature EMAP II;

[0028]FIG. 2 illustrates the effects of human full-length TyrRS, humanmini TyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAPII, and E. coli TyrRS on MP chemotaxis (white bars) and MP TNFαproduction (gray bars);

[0029]FIG. 3 illustrates the effects of human full-length TyrRS, humanmini TyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAPII, and E. coli TyrRS on MP tissue factor production (white bars) andPMN release of myeloperoxidase (gray bars);

[0030]FIG. 4 illustrates the effects of human full-length TyrRS, humanmini TyrRS, the human TyrRS carboxyl-terminal domain, human mature EMAPII, E. coli TyrRS, human TyrRS mutant, and IL-8 on PMN chemotaxis;

[0031]FIG. 5 illustrates a schematic partial alignment of human miniTyrRS (SEQ ID NO:17), E. coli TyrRS (SEQ ID NO:18), IL-8 (SEQ ID NO:19),Groα (SEQ ID NO:20), and NAP-2 (SEQ ID NO:21); the connectivepolypeptide 1 (CP1) which splits the Rossmann nucleotide-binding fold inTyrRS is indicated;

[0032]FIG. 6 illustrates the results of competition assays in whichunlabeled human mini TyrRS, human TyrRS mutant, human full-length TyrRS,the human TyrRS carboxyl-terminal domain, human mature EMAP II, E. coliTyrRS, IL-8, Groα, or NAP-2 was used in molar excess with ¹²⁵I-humanmini TyrRS on PMNs;

[0033] FIGS. 7A-7B illustrate the results of immunoblot analysis of (A)supernatants following growth of U-937 cells in normal media (Lane 1)and serum-free media (Lane 2), and (B) supernatants following growth ofU-937 cells in serum-free media for 4, 12, or 24 hours (Lanes 1-3), orfrom a cell extract isolated from U-937 cells grown in serum-free mediafor 24 hours;

[0034] FIGS. 8A-8B illustrate (A) the results of immunoblot analysis ofhuman full-length TyrRS (Lane 1), human mini TyrRS (Lane 2), the TyrRScarboxyl-terminal domain (Lane 3), the extended carboxyl-terminal domainof human TyrRS (Lane 4), and human full-length TyrRS following cleavagewith PMN elastase (Lane 5), and (B) a schematic representation of thecleavage sites for human pro-EMAP II (SEQ ID NO:22) and humanfull-length TyrRS (SEQ ID NO:23);

[0035]FIG. 9 illustrates a schematic alignment of human full-lengthTyrRS, human mini TyrRS, S. cerevisiae TyrRS, and E. coli TyrRS;

[0036]FIG. 10 illustrates the results of competition assays in whichunlabeled human mini TyrRS, S. cerevisiae TyrRS, or E. coli TyrRS wasused in molar excess with ¹²⁵I-human mini TyrRS on PMNs;

[0037]FIG. 11 illustrates the effects of human mini TyrRS, S. cerevisiaeTyrRS, or E. coli TyrRS on PMN chemotaxis;

[0038]FIG. 12 illustrates a schematic alignment of the human EMAPII-like domains and corresponding synthetic peptides for humanfull-length TyrRS (SEQ ID NO:24), human EMAP II (SEQ ID NO:25-26), C.elegans MetRS (SEQ ID NO:27), and S. cervisiae Arc1p (SEQ ID NO:28);

[0039]FIG. 13 illustrates the effects of synthetic peptides derived fromhuman TyrRS, human EMAP II, C. elegans MetRS, and S. cervisiae Arc1p onPMN chemotaxis

[0040]FIG. 14 illustrates a schematic comparison between human miniTyrRS and α-chemokines (the carboxyl-terminal portions of each have beenomitted); the location of β-sheets (solid arrows) and ELR motifs(circles) are indicated;

[0041]FIG. 15 illustrates a schematic alignment of human full-lengthTyrRS, human mini TyrRS, human full-length TrpRS, and human mini TrpRS;the carboxyl-terminal and amino-terminal appended domains (hatched) areindicated;

[0042]FIG. 16 illustrates the angiogenic activity of human full-lengthTyrRS, human mini TyrRS, human mini TyrRS mutant, or human VEGF on HUVECchemotaxis;

[0043]FIG. 17 illustrates the angiogenic activity of human full-lengthTyrRS, human mini TyrRS, human mini TyrRS mutant, human miniTyrRS+IP-10, human VEGF, or human VEGF+IP-10 on the CAMs of 10-day-oldchick embryos;

[0044]FIG. 18 illustrates the angiogenic activity of human full-lengthTyrRS or human mini TyrRS on endothelial cell proliferation;

[0045]FIG. 19 illustrates the effect of human full-length TrpRS or humanmini TrpRS on human VEGF-induced or human mini TyrRS-induced endothelialmigration;

[0046]FIG. 20 illustrates the effect of human full-length TrpRS or humanmini TrpRS on the angiogenic activity of human VEGF or human mini TyrRSon chick CAM;

[0047]FIG. 21 illustrates the results of immunoblot analysis showing thesecretion of human TrpRS from human U-937 cells;

[0048]FIG. 22 illustrates the cleavage of human full-length TrpRS by PMNelastase; TrpRS was exposed to PMN elastase for 0 minutes (Lane 1), 15minutes (Lane 2), 30 minutes (Lane 3), or 60 minutes (Lane 4);

[0049]FIG. 23 illustrates the effect of human supermini TrpRS on theangiogenic activity of human VEGF or human mini TyrRS on chick CAM;

[0050]FIG. 24 illustrates the amino acid sequence similarity of humanTrpRS (SEQ ID NO:45 and SEQ ID NO:51), bovine TrpRS (SEQ ID NO:46 andSEQ ID NO:52), mouse TrpRS (SEQ ID NO:47 and SEQ ID NO:53), rabbit TrpRS(SEQ ID NO:48 and SEQ ID NO:54), human semaphorin-E (SEQ ID NO:49),mouse semaphorin-E (SEQ ID NO:50), and mouse neuropilin-2 (SEQ ID NO:55); the amino-terminal residue of the mini, supermini, and inactiveforms of TrpRS (arrows), identical residues (asterisks), semi-conservedresidues (dots), and insertions in the c-domain of neurophilin-2 (bars)are indicated;

[0051]FIG. 25 illustrates the regions of semaphorin-E and neuropilin-2that share sequence similarity with mammalian TrpRS (indicated byarrows); semaphorin-E and neuropilin-2 domains are indicated as follows:semaphorin domain (sema), immunoglobulin domain (Ig), carboxy-terminalbasic domain (C), complement-binding domain (a1, a2), coagulation factordomain (b1, b2), c domain (c), transmembrane domain (TM), andcytoplasmic domain (Cy). Isoforms of neuropilin exist with insertions of5, 17, or 22 amino acids in the c-domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Definitions

[0053] “Truncated tRNA synthetase polypeptides” means polypeptides thatare shorter than the corresponding full length tRNA synthetase.

[0054] “TrpRS” means tryptophanyl-tRNA synthetase.

[0055] “TyrRS” means tyrosyl-tRNA synthetase.

[0056] “Cell culture” encompasses both the culture medium and thecultured cells.

[0057] The phrase “isolating a polypeptide from the cell culture”encompasses isolating a soluble or secreted polypeptide from the culturemedium as well as isolating an integral membrane protein from thecultured cells.

[0058] “Cell extract” includes culture media, especially spent culturemedia from which the cells have been removed. A cell extract thatcontains the DNA or protein of interest should be understood to mean ahomogenate preparation or cell-free preparation obtained from cells thatexpress the protein or contain the DNA of interest.

[0059] “Plasmid” is an autonomous, self-replicating extrachromosomal DNAmolecule and is designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0060] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 degrees C. are ordinarily used, but may vary in accordancewith the supplier's instructions. After digestion the reaction iselectrophoresed directly on a poly-acrylamide gel to isolate the desiredfragment. The nucleotides present in various DNA and RNA fragments aredesignated herein by the standard single letter designations (A, T, C,G, U) used in the art.

[0061] “Polynucleotide” of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in SEQ ID NO:1 or in SEQ ID NO:9or may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide sequence shown in SEQ ID NO:1 or in SEQ ID NO:9.

[0062] The term “Polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

[0063] “Oligonucleotides” refers to either a single strandedpolynucleotide or two complementary polynucleotide strands which may bechemically synthesized. Such synthetic oligonucleotides have no 5′phosphate and thus will not ligate to another oligonucleotide withoutadding a phosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0064] “Amino acid residue” refers to an amino acid which is part of apolypeptide. The amino acid residues described herein are preferably inthe L″ isomeric form. However, residues in the D″ isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxyl terminus of apolypeptide. In keeping with standard polypeptide nomenclature describedin J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 C.F.R.§§1.821-1.822, abbreviations for amino acid residues are shown in thefollowing Table: TABLE 1 Table of Correspondence SYMBOL 1-Letter3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine MMet methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucineT Thr threonine V Val valine P Pro proline K Lys lysine H His histidineQ Gln glutamine E Glu glutamic acid Z Glx Glu and/or Gln W Trptryptophan R Arg arginine D Asp aspartic acid N Asn asparagine B Asx Asnand/or Asp C Cys cysteine X Xaa Unknown or other

[0065] All amino acid residue sequences represented herein by formulaehave a left to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is broadly defined to include the amino acids listed in Table 1as well as modified and unusual amino acids, such as those referred toin 37 C.F.R. §§1.821-1.822, and incorporated herein by reference. A dashat the beginning or end of an amino acid residue sequence indicates apeptide bond to a further sequence of one or more amino acid residues orto an amino-terminal group such as NH₂ or to a carboxyl-terminal groupsuch as COOH.

[0066] In a peptide or protein, suitable conservative substitutions ofamino acids are known to those of skill in this art and may be madegenerally without altering the biological activity of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/CummingsPub. co., p.224).

[0067] Such substitutions are preferably made in accordance with thoseset forth in TABLE 2 as follows: TABLE 2 Original residue Conservativesubstitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) SerGln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu;Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F)Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val(V) Ile; Leu

[0068] Other substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions.

[0069] “Complementing plasmid” describes plasmid vectors that delivernucleic acids into a packaging cell line for stable integration into achromosome in the cellular genome.

[0070] “Delivery plasmid” is a plasmid vector that carries or deliversnucleic acids encoding a therapeutic gene or gene that encodes atherapeutic product or a precursor thereof or a regulatory gene or otherfactor that results in a therapeutic effect when delivered in vivo in orinto a cell line, such as, but not limited to a packaging cell line, topropagate therapeutic viral vectors.

[0071] A variety of vectors is described herein. For example, one vectoris used to deliver particular nucleic acid molecules into a packagingcell line for stable integration into a chromosome. These types ofvectors are generally identified herein as complementing plasmids. Afurther type of vector described herein carries or delivers nucleic acidmolecules in or into a cell line (e.g., a packaging cell line) for thepurpose of propagating therapeutic viral vectors; hence, these vectorsare generally referred to herein as delivery plasmids. A third “type” ofvector described herein is used to carry nucleic acid molecules encodingtherapeutic proteins or polypeptides or regulatory proteins or areregulatory sequences to specific cells or cell types in a subject inneed of treatment; these vectors are generally identified herein astherapeutic viral vectors or recombinant adenoviral vectors or viralAd-derived vectors and are in the form of a virus particle encapsulatinga viral nucleic acid containing an expression cassette for expressingthe therapeutic gene.

[0072] “DNA or nucleic acid homolog” refers to a nucleic acid thatincludes a preselected conserved nucleotide sequence, such as a sequenceencoding a therapeutic polypeptide. By the term “substantiallyhomologous” is meant having at least 80%, preferably at least 90%, mostpreferably at least 95% homology therewith or a less percentage ofhomology or identity and conserved biological activity or function.

[0073] The terms “homology” and “identity” are often usedinterchangeably. In this regard; degree of homology or identity may bedetermined, for example, by comparing sequence information using a GAPcomputer program. The GAP program utilizes the alignment method ofNeedleman and Wunsch (J. Mol. Biol. 48:443 (1970), as revised by Smithand Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP programdefines similarity as the number of aligned symbols (i.e., nucleotidesor amino acids) which are similar, divided by the total number ofsymbols in the shorter of the two sequences. The preferred defaultparameters for the GAP program may include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix of Gribskov and Burgess, Nucl. AcidsRes. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLASOF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical ResearchFoundation, pp.353-358 (1979); (2) a penalty of 3.0 for each gap and anadditional 0.10 penalty for each symbol in each gap; and (3) no penaltyfor end gaps. Whether any two nucleic acid molecules have nucleotidesequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical” can be determined using known computer algorithms such asthe “FAST A” program, using for example, the default parameters as inPearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988).Alternatively the BLAST function of the National Center forBiotechnology Information database may be used to determine identity. Ingeneral, sequences are aligned so that the highest order match isobtained. “Identity” per se has an art-recognized meaning and can becalculated using published techniques. (See, e.g.: ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity between two polynucleotide or polypeptidesequences, the term “identity” is well known to skilled artisans(Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)). Methodscommonly employed to determine identity or similarity between twosequences include, but are not limited to, those disclosed in Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988).Methods to determine identity and similarity are codified in computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(I):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol215:403 (1990)).

[0074] The term “identity” represents a comparison between a test and areference polypeptide or polynucleotide. For example, a test polypeptidemay be defined as any polypeptide that is 90% or more identical to areference polypeptide. As used herein, the term at least “90% identicalto” refers to percent identities from 90 to 99.99 relative to thereference polypeptides. Identity at a level of 90% or more is indicativeof the fact that, assuming for exemplification purposes a test andreference polynucleotide length of 100 amino acids are compared. No morethan 10% (i.e., 10 out of 100) amino acids in the test polypeptidediffers from that of the reference polypeptides. Similar comparisons maybe made between a test and reference polynucleotides. Such differencesmay be represented as point mutations randomly distributed over theentire length of an amino acid sequence or they may be clustered in oneor more locations of varying length up to the maximum allowable, e.g.10/100 amino acid difference (approximately 90% identity). Differencesare defined as nucleic acid or amino acid substitutions, or deletions.

[0075] The terms “gene therapy” and “genetic therapy” refer to thetransfer of heterologous DNA to the certain cells, target cells, of amammal, particularly a human, with a disorder or conditions for whichsuch therapy is sought. The DNA is introduced into the selected targetcells in a manner such that the heterologous DNA is expressed and atherapeutic product encoded thereby is produced. Alternatively, theheterologous DNA may in some manner mediate expression of DNA thatencodes the therapeutic product, it may encode a product, such as apeptide or RNA that in some manner mediates, directly or indirectly,expression of a therapeutic product. Genetic therapy may also be used tonucleic acid encoding a gene product replace a defective gene orsupplement a gene product produced by the mammal or the cell in which itis introduced. The introduced nucleic acid may encode a therapeuticcompound, such as a growth factor inhibitor thereof, or a tumor necrosisfactor or inhibitor thereof, such as a receptor therefor, that is notnormally produced in the mammalian host or that is not produced intherapeutically effective amounts or at a therapeutically useful time.The heterologous DNA encoding the therapeutic product may be modifiedprior to introduction into the cells of the afflicted host in order toenhance or otherwise alter the product or expression thereof.

[0076] “Heterologous DNA” is DNA that encodes RNA and proteins that arenot normally produced in vivo by the cell in which it is expressed orthat mediates or encodes mediators that alter expression of endogenousDNA by affecting transcription, translation, or other regulatablebiochemical processes. Heterologous DNA may also be referred to asforeign DNA. Any DNA that one of skill in the art would recognize orconsider as heterologous or foreign to the cell in which is expressed isherein encompassed by heterologous DNA. Examples of heterologous DNAinclude, but are not limited to, DNA that encodes traceable markerproteins, such as a protein that confers drug resistance, DNA thatencodes therapeutically effective substances, such as anti-canceragents, enzymes and hormones, and DNA that encodes other types ofproteins, such as antibodies. Antibodies that are encoded byheterologous DNA may be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced. Hence, “heterologousDNA” or “foreign DNA”, refers to a DNA molecule not present in the exactorientation and position as the counterpart DNA molecule found in thecorresponding wild-type adenovirus. It may also refer to a DNA moleculefrom another organism or species (i.e., exogenous) or from another Adserotype.

[0077] “Therapeutically effective DNA product” is a product that isencoded by heterologous DNA so that, upon introduction of the DNA into ahost, a product is expressed that effectively ameliorates or eliminatesthe symptoms, manifestations of an inherited or acquired disease or thatcures said disease. Typically, DNA encoding the desired heterologous DNAis cloned into a plasmid vector and introduced by routine methods, suchas calcium-phosphate mediated DNA uptake (see, (1981) Somat. Cell. Mol.Genet. 7:603-616) or micro injection, into producer cells, such aspackaging cells. After amplification in producer cells, the vectors thatcontain the heterologous DNA are introduced into selected target cells.

[0078] “Expression or delivery vector” refers to any plasmid or virusinto which a foreign or heterologous DNA may be inserted for expressionin a suitable host cell—i.e., the protein or polypeptide encoded by theDNA is synthesized in the host cell's system. Vectors capable ofdirecting the expression of DNA segments (genes) encoding one or moreproteins are referred to herein as “expression vectors.” Also includedare vectors that allow cloning of cDNA (complementary DNA) from mRNAsproduced using reverse transcriptase.

[0079] “Gene” is a nucleic acid molecule whose nucleotide sequenceencodes RNA or polypeptide. A gene can be either RNA or DNA. Genes mayinclude regions preceding and following the coding region (leader andtrailer) as well as intervening sequences (introns) between individualcoding segments (exons).

[0080] “Isolated” with reference to a nucleic acid molecule,polypeptide, or other biomolecule, means that the nucleic acid orpolypeptide has separated from the genetic environment from which thepolypeptide or nucleic acid were obtained. It may also mean altered fromthe natural state. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Thus, apolypeptide or polynucleotide produced and/or contained within arecombinant host cell is considered isolated. Also intended as an“isolated polypeptide” or an “isolated polynucleotide” are polypeptidesor polynucleotides that have been purified, partially or substantially,from a recombinant host cell or from a native source. For example, arecombinantly produced version of a compounds can be substantiallypurified by the one-step method described in Smith and Johnson, Gene67:31-40 (1988). The terms “isolated” and “purified” are sometimes usedinterchangeably. Such polynucleotide could be part of a vector and/orsuch polynucleotide or polypeptide could be part of a composition, andstill be isolated in that such vector or composition is not part of itsnatural environment.

[0081] By “isolated polynucleotide” is meant that the nucleic acid isfree of the coding sequences of those genes that, in thenaturally-occurring genome of the organism (if any) immediately flankthe gene encoding the nucleic acid of interest. Isolated DNA may besingle-stranded or double-stranded, and may be genomic DNA, cDNA,recombinant hybrid DNA, or synthetic DNA. It may be identical to anative DNA sequence, or may differ from such sequence by the deletion,addition, or substitution of one or more nucleotides.

[0082] “Isolated” or “purified” as it refers to preparations made frombiological cells or hosts means any cell extract containing theindicated DNA or protein including a crude extract of the DNA or proteinof interest. For example, in the case of a protein, a purifiedpreparation can be obtained following an individual technique or aseries of preparative or biochemical techniques and the DNA or proteinof interest can be present at various degrees of purity in thesepreparations. The procedures may include for example, but are notlimited to, ammonium sulfate fractionation, gel filtration, ion exchangechange chromatography, affinity chromatography, density gradientcentrifugation and electrophoresis.

[0083] A preparation of DNA or protein that is “substantially pure” or“isolated” means a preparation free from naturally occurring materialswith which such DNA or protein is normally associated in nature.“Essentially pure” should be understood to mean a “highly” purifiedpreparation that contains at least 95% of the DNA or protein ofinterest.

[0084] “Packaging cell line” is a cell line that provides a missing geneproduct or its equivalent.

[0085] “Adenovirus viral particle” is the minimal structural orfunctional unit of a virus. A virus can refer to a single particle, astock of particles or a viral genome. The adenovirus (Ad) particle isrelatively complex and may be resolved into various substructures.

[0086] “Post-transcription regulatory element (PRE)” is a regulatoryelement found in viral or cellular messenger RNA that is not spliced,i.e. intronless messages. Examples include, but are not limited to,human hepatitis virus, woodchuck hepatitis virus, the TK gene and mousehistone gene. The PRE may be placed before a polyA sequence and after aheterologous DNA sequence.

[0087] “Pseudo typing” describes the production of adenoviral vectorshaving modified capsid protein or capsid proteins from a differentserotype than the serotype of the vector itself. One example, is theproduction of an adenovirus 5 vector particle containing an Ad37 fiberprotein. This may be accomplished by producing the adenoviral vector inpackaging cell lines expressing different fiber proteins.

[0088] “Promoters of interest herein” may be inducible or constitutive.Inducible promoters will initiate transcription only in the presence ofan additional molecule; constitutive promoters do not require thepresence of any additional molecule to regulate gene expression. Aregulatable or inducible promoter may also be described as a promoterwhere the rate or extent of RNA polymerase binding and initiation ismodulated by external stimuli. Such stimuli include, but are not limitedto various compounds or compositions, light, heat, stress and chemicalenergy sources. Inducible, suppressible and repressible promoters areconsidered regulatable promoters. Preferred promoters herein, arepromoters that are selectively expressed in ocular cells, particularlyphoto receptor cells.

[0089] “Receptor” refers to a biologically active molecule thatspecifically binds to (or with) other molecules. The term “receptorprotein” may be used to more specifically indicate the proteinaceousnature of a specific receptor.

[0090] “Recombinant” refers to any progeny formed as the result ofgenetic engineering. This may also be used to describe a virus formed byrecombination of plasmids in a packaging cell.

[0091] “Transgene” or “therapeutic nucleic acid molecule” includes DNAand RNA molecules encoding an RNA or polypeptide. Such molecules may be“native” or naturally-derived sequences; they may also be “non-native”or “foreign” that are naturally- or recombinantly-derived. The term“transgene,” which may be used interchangeably herein with the term“therapeutic nucleic acid molecule,” is often used to describe aheterologous or foreign (exogenous) gene that is carried by a viralvector and transduced into a host cell. Therapeutic nucleotide nucleicacid molecules include antisense sequences or nucleotide sequences whichmay be transcribed into antisense sequences. Therapeutic nucleotidesequences (or transgenes) all include nucleic acid molecules thatfunction to produce a desired effect in the cell or cell nucleus intowhich said therapeutic sequences are delivered. For example, atherapeutic nucleic acid molecule can include a sequence of nucleotidesthat encodes a functional protein intended for delivery into a cellwhich is unable to produce that functional protein.

[0092] “Vitreous of the eye” refers to a material that fills the chamberbehind the lens of the eye (i.e., vitreous humor or vitreous body).

[0093] “Promoter region” refers to the portion of DNA of a gene thatcontrols transcription of the DNA to which it is operatively linked. Thepromoter region includes specific sequences of DNA that are sufficientfor RNA polymerase recognition, binding and transcription initiation.This portion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of the RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. “Operatively linked” meansthat the sequences or segments have been covalently joined into onepiece of DNA, whether in single or double stranded form, whereby controlsequences on one segment control expression or replication or other suchcontrol of other segments. The two segments are not necessarilycontiguous, however.

[0094] “Package” refers to a solid matrix or material such as glass,plastic (e.g., polyethylene, polypropylene or polycarbonate), paper,foil and the like capable of holding within fixed limits a polypeptide,polyclonal antibody, or monoclonal antibody of the present invention.Thus, for example, a package can be a glass vial used to containmilligram quantities of a contemplated polypeptide or it can be a microtiter plate well to which microgram quantities of a contemplatedpolypeptide or antibody have been operatively affixed (i.e., linked) soas to be capable of being immunologically bound by an antibody orantigen, respectively.

[0095] “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

[0096] “Diagnostic system” in the context of the present invention alsoincludes a label or indicating means capable of signaling the formationof an immunocomplex containing a polypeptide or antibody molecule of thepresent invention.

[0097] “Complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

[0098] “Label” and “Indicating means” in their various grammatical formsrefer to single atoms and molecules that are either directly orindirectly involved in the production of a detectable signal to indicatethe presence of a complex. Any label or indicating means can be linkedto or incorporated in an expressed protein, polypeptide, or antibodymolecule that is part of an antibody or monoclonal antibody compositionof the present invention or used separately, and those atoms ormolecules can be used alone or in conjunction with additional reagents.Such labels are themselves well-known in clinical diagnostic chemistryand constitute a part of this invention only insofar as they areutilized with otherwise novel proteins methods and/or systems.

[0099] The terms “fragment,” “derivative” and “analog”, when referringto a polypeptide, means a polypeptide which retains substantially thesame biological function or activity as such polypeptide. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0100] A “specific binding agent” is a molecular entity capable ofselectively binding a reagent species of the present invention or acomplex containing such a species, but is not itself a polypeptide orantibody molecule composition of the present invention. Exemplaryspecific binding agents are second antibody molecules, complementproteins or fragments thereof, S. aureus protein A, and the like.

[0101] “ELISA” refers to an enzyme-linked immunosorbent assay thatemploys an antibody or antigen bound to a solid phase and anenzyme-antigen or enzyme-antibody conjugate to detect and quantify theamount of an antigen present in a sample. A description of the ELISAtechnique is found in Sites et al., Basic and Clinical Immunology,4^(th) Ed., Chap. 22, Lange Medical Publications, Los Altos, Calif.(1982) and in U.S. Pat. No. 3,654,090; U.S. Pat. No. 3,850,752; and U.S.Pat. No. 4,016,043, which are all incorporated herein by reference.

[0102] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the amino acidsequence of SEQ ID NO:9 or the polypeptide encoded by the cDNA of SEQ IDNO:9. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide. Thus, the present invention includespolynucleotides encoding the same mature polypeptide as shown SEQ IDNO:9 or the same mature polypeptide encoded by the cDNA of SEQ ID NO:9as well as variants of such polynucleotides which variants encode for anfragment, derivative or analog of the polypeptide of SEQ ID NO:9 or thepolypeptide encoded by SEQ ID NO:9. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

[0103] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in SEQ ID NO:1 or SEQ ID NO:9. As known in the art, an“allelic variant” is an alternate form of a polynucleotide sequencewhich have a substitution, deletion or addition of one or morenucleotides, which does not substantially alter the function of theencoded polypeptide.

[0104] Thus, for example, the poynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and prosequence (leadersequence).

[0105] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markdersequence may abe a hexahistidine tag supplied be a pQE-9 vector toprovide for purificaation of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemmagglutinin (HA) tag when a mimalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0106] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least50% and preferably 70% identity between the sequences. The presentinvention particularly relates to polynucleotides which hybridize understringent conditions to the hereinabove-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which retain substantially the same biological function oractivity as the mature polypeptide encoded by the cDNA of SEQ ID NO:9.

[0107] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0108] The fragment, derivative or analog of the polypeptide of SEQ IDNO:9 or that encoded by the polynucleotide of SEQ ID NO:9 may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0109] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0110] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0111] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the tRNA synthetase polpeptide genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0112] The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudo rabies. A preferred vector is pET20b. However, any otherplasmid or vector may be used as long as it is replicable and viable inthe host.

[0113] As hereinabove described, the appropriate DNA sequence may beinserted into the vector by a variety of procedures. In general, the DNAsequence is inserted into an appropriate restriction endonuclease sitesby procedures known in the art. Such procedures and others are deemed tobe within the scope of those skilled in the art.

[0114] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L)promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0115] In addition, the expression vectors preferably contain a gene toprovide a phenotypic trait for selection of transformed host cells suchas dihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

[0116] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence,may be employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium, Streptomyces; fungal cells, such as yeast; insect cells,such as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

[0117] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE-9 (Qiagen), pBs,phage script, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, PRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, PMSG, pSVL (Pharmacia) and pET20B. In one preferredembodiment, the vector is pET20B. However, any other plasmid or vectormay be used as long as they are replicable and viable in the host.

[0118] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), PLand trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0119] In a further embodiment, the present invention relates to hostcells containing the above-described construct. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, 1986)).

[0120] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0121] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook. et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), the disclosure of which is hereby incorporated byreference.

[0122] Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

[0123] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha.-factor, acid phosphatase, orheat shock proteins, among others. The heterologous structural sequenceis assembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0124] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0125] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0126] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

[0127] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0128] Polypeptides are recovered and purified from recombinant cellcultures by methods used heretofore, including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxyapatite chromatographyand lectin chromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price, et al., J. Biol. Chem., 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

[0129] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated.

[0130] The polypeptides of the present invention may be modified toimprove stability and increase potency by means known in the art. Forexample, L-amino acids can be replaced by D-amino acids, the aminoterminus can be acetylated, or the carboxyl terminus modified, e.g.,ethyl amine-capped (Dawson, D. W., et al., Mol. Pharmacol., 55: 332-338(1999)).

[0131] Angiogenic tRNA synthetase polypeptides are useful as woundhealing agents, particularly where it is necessary to re-vascularizedamaged tissues, or where new capillary angiogenesis is important.Therefore, it may be used for treatment of full-thickness wounds such asdermal ulcers, including pressure sores, venous ulcers, and diabeticulcers. In addition, it can be used in the treatment of full-thicknessburns and injuries where angiogenesis is desired to prepare the burn ininjured sites for a skin graft and flap. In this case, it should beapplied directly at the sites. Similarly angiogenic TRNA synthetasepolypeptides polypeptides can be used in plastic surgery whenreconstruction is required following a burn, other trauma, or even forcosmetic purposes.

[0132] Since angiogenesis is important in keeping wounds clean andnon-infected, angiogenic tRNA synthetase polypeptides may be used inassociation with surgery and following the repair of cuts. It should beparticularly useful in the treatment of abdominal wounds where there isa high risk of infection.

[0133] Angiogenic tRNA synthetase polypeptides can be used for thepromotion of endothelialization in vascular graft surgery. In the caseof vascular grafts using either transplanted or synthetic material,angiogenic tyrRS peptides can be applied to the surface of the graft,preferably in a pharmaceutically appropriate excipient. In oneembodiment, the pharmaceutically appropriate excipient further providesfor the continuous release of angiogenic tyrRS peptides.

[0134] Angiogenic tRNA synthetase therapy can be used to repair thedamage of myocardial infarction. In one preferred embodiment, angiogenictyrRS therapy can be used in conjunction with coronary bypass surgery bystimulating the growth of the transplanted tissue. In one preferredembodiment, angiogenic tRNA synthetase therapy can be administered bydirect myocardial injection of angiogenic tRNA synthetase polypeptidesor polynucleotides encoding angiogenic tRNA synthetase polypeptides. SeeLosodo, D. W., et al., Circulation, 98: 2800-2804 (1998).

[0135] In another embodiment, angiogenic tRNA synthetase therapy can beused in conjunction with angiography to administer the angiogenic tRNAsynthetase polypeptides or polynucleotides encoding angiogenic tRNAsynthetase polypeptides directly to the lumen and wall of the bloodvessel.

[0136] Similarly, tRNA synthetase therapy can be used to administer theangiogenic or angiostatic TRNA synthetase polypeptides orpolynucleotides encoding angiogenic or angiostatic tRNA synthetasepolypeptides directly to the lumen and wall of other hollow organs, suchas the uterus.

[0137] The polypeptide of the present invention may also be employed inaccordance with the present invention by expression of such polypeptidein vivo, which is often referred to as “gene therapy.”

[0138] Thus, for example, cells such as bone marrow cells may beengineered with a polynucleotide (DNA or RNA) encoding for thepolypeptide ex vivo, the engineered cells are then provided to a patientto be treated with the polypeptide. Such methods are well-known in theart. For example, cells may be engineered by procedures known in the artby use of a retroviral particle containing RNA encoding for thepolypeptide of the present invention. Similarly, cells may be engineeredin vivo for expression of the polypeptide in vivo, for example, byprocedures known in the art. As known in the art, a producer cell forproducing a retroviral particle containing RNA encoding the polypeptideof the present invention may be administered to a patient forengineering cells in vivo and expression of the polypeptide in vivo.These and other methods for administering a polypeptide of the presentinvention by such methods should be apparent to those skilled in the artfrom the teachings of the present invention. For example, the expressionvehicle for engineering cells may be other than a retroviral particle,for example, an adenovirus, which may be used to engineering cells invivo after combination with a suitable delivery vehicle.

[0139] Various viral vectors that can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia,adeno-associated virus (AAV), or, preferably, an RNA virus such as aretrovirus. Preferably, the retroviral vector is a derivative of amurine or avian retrovirus, or is a lentiviral vector. The preferredretroviral vector is a lentiviral vector. Examples of retroviral vectorsin which a single foreign gene can be inserted include, but are notlimited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV,HIV and Rous Sarcoma Virus (RSV). A number of additional retroviralvectors can incorporate multiple genes. All of these vectors cantransfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a zincfinger derived-DNA binding polypeptide sequence of interest into theviral vector, along with another gene that encodes the ligand for areceptor on a specific target cell, for example, the vector is madetarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome to allow target specificdelivery of the retroviral vector containing the zinc finger-nucleotidebinding protein polynucleotide.

[0140] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsitation. Helper cell lineswhich have deletions of the packaging signal include but are not limitedto Ψ2, PA317 and PA12, for example. These cell lines produce emptyvirions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced. The vector virions producedby this method can then be used to infect a tissue cell line, such asNIH 3T3 cells, to produce large quantities of chimeric retroviralvirions.

[0141] Another targeted delivery system for polynucleotides encodingzinc finger derived-DNA binding polypeptides is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, micro spheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles which are useful as delivery vehiclesin vitro and in vivo. It has been shown that large unilamellar vesicles(LV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

[0142] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0143] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0144] The targeting of liposomes has been classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specific,and organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0145] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. In general, the compounds bound tothe surface of the targeted delivery system will be ligands andreceptors which will allow the targeted delivery system to find and“home in” on the desired cells. A ligand may be any compound of interestwhich will bind to another compound, such as a receptor.

[0146] In general, surface membrane proteins which bind to specificeffector molecules are referred to as receptors. In the presentinvention, antibodies are preferred receptors. Antibodies can be used totarget liposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), may be exploited for the purpose oftargeting antibody-zinc finger-nucleotide binding protein-containingliposomes directly to the malignant tumor. Since the zincfinger-nucleotide binding protein gene product may be indiscriminatewith respect to cell type in its action, a targeted delivery systemoffers a significant improvement over randomly injecting non-specificliposomes. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an the antigenic epitope on the target cells.Liposomes may also be targeted to cells expressing receptors forhormones or other serum factors.

[0147] There are available to one skilled in the art multiple viral andnon-viral methods suitable for introduction of a nucleic acid moleculeinto a target cell. Genetic manipulation of primary tumor cells has beendescribed previously (Patel et al., 1994). Genetic modification of acell may be accomplished using one or more techniques well known in thegene therapy field (Human Gene Therapy, April 1994, Vol. 5, p. 543-563;Mulligan, R. C. 1993). Viral transduction methods may comprise the useof a recombinant DNA or an RNA virus comprising a nucleic acid sequencethat drives or inhibits expression of a protein having sialyltransferaseactivity to infect a target cell. A suitable DNA virus for use in thepresent invention includes but is not limited to an adenovirus (Ad),adeno-associated virus (AAV), herpes virus, vaccinia virus or a poliovirus. A suitable RNA virus for use in the present invention includesbut is not limited to a retrovirus or Sindbis virus. It is to beunderstood by those skilled in the art that several such DNA and RNAviruses exist that may be suitable for use in the present invention.

[0148] Adenoviral vectors have proven especially useful for genetransfer into eukaryotic cells (Stratford-Perricaudet and Perricaudet.1991). Adenoviral vectors have been successfully utilized to studyeukaryotic gene expression (Levrero, M., et al. 1991). vaccinedevelopment (Graham and Prevec, 1992), and in animal models(Stratford-Perricaudet, et al. 1992.; Rich, et al. 1993). The firsttrial of Ad-mediated gene therapy in human was the transfer of thecystic fibrosis transmembrane conductance regulator (CFTR) gene to lung(Crystal, et al., 1994). Experimental routes for administratingrecombinant Ad to different tissues in vivo have included intra trachealinstillation (Rosenfeld, et al. 1992) injection into muscle (Quantin,B., et al. 1992), peripheral intravenous injection (Herz and Gerard,1993) and stereotactic inoculation to brain (Le Gal La Salle, et al.1993). The adenoviral vector, then, is widely available to one skilledin the art and is suitable for use in the present invention.

[0149] Adeno-associated virus (AAV) has recently been introduced as agene transfer system with potential applications in gene therapy.Wild-type AAV demonstrates high-level infectivity, broad host range andspecificity in integrating into the host cell genome (Hermonat andMuzyczka. 1984). Herpes simplex virus type-1 (HSV-1) is attractive as avector system, especially for use in the nervous system because of itsneurotropic property (Geller and Federoff. 1991; Glorioso, et al. 1995).Vaccinia virus, of the poxvirus family, has also been developed as anexpression vector (Smith and Moss, 1983; Moss, 1992). Each of theabove-described vectors are widely available to one skilled in the artand would be suitable for use in the present invention.

[0150] Retroviral vectors are capable of infecting a large percentage ofthe target cells and integrating into the cell genome (Miller andRossmann. 1989). Retroviruses were developed as gene transfer vectorsrelatively earlier than other viruses, and were first used successfullyfor gene marking and transducing the cDNA of adenosine deaminase (ADA)into human lymphocytes. Preferred retroviruses include lentiviruses. Inpreferred embodiments, the retrovirus is selected from the groupconsisting of HIV, BIV and SIV.

[0151] “Non-viral” delivery techniques that have been used or proposedfor gene therapy include DNA-ligand complexes, adenovirus-ligand-DNAcomplexes, direct injection of DNA, CaPO₄ precipitation, gene guntechniques, electroporation, liposomes and lipofection (Mulligan, 1993).Any of these methods are widely available to one skilled in the art andwould be suitable for use in the present invention. Other suitablemethods are available to one skilled in the art, and it is to beunderstood that the present invention may be accomplished using any ofthe available methods of transfection. Several such methodologies havebeen utilized by those skilled in the art with varying success(Mulligan, R. 1993). Lipofection may be accomplished by encapsulating anisolated DNA molecule within a liposomal particle and contacting theliposomal particle with the cell membrane of the target cell. Liposomesare self-assembling, colloidal particles in which a lipid bilayer,composed of amphiphilic molecules such as phosphatidyl serine orphosphatidyl choline, encapsulates a portion of the surrounding mediasuch that the lipid bilayer surrounds a hydrophilic interior.unilamellar or multilamellar liposomes can be constructed such that theinterior contains a desired chemical, drug, or, as in the instantinvention, an isolated DNA molecule.

[0152] The cells may be transfected in vivo, ex vivo, or in vitro. Thecells may be transfected as primary cells isolated from a patient or acell line derived from primary cells, and are not necessarily autologousto the patient to whom the cells are ultimately administered. Followingex vivo or in vitro transfection, the cells may be implanted into ahost. Genetic manipulation of primary tumor cells has been describedpreviously (Patel et al. 1994). Genetic modification of the cells may beaccomplished using one or more techniques well known in the gene therapyfield (Human Gene Therapy. April 1994. Vol. 5, p. 543-563; Mulligan, R.C. 1993).

[0153] In order to obtain transcription of the nucleic acid of thepresent invention within a target cell, a transcriptional regulatoryregion capable of driving gene expression in the target cell isutilized. The transcriptional regulatory region may comprise a promoter,enhancer, silencer or repressor element and is functionally associatedwith a nucleic acid of the present invention. Preferably, thetranscriptional regulatory region drives high level gene expression inthe target cell. Transcriptional regulatory regions suitable for use inthe present invention include but are not limited to the humancytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 earlyenhancer/promoter, the JC polyomavirus promoter, the albumin promoter,PGK and the a-actin promoter coupled to the CMV enhancer (Doll, et al.1996).

[0154] The vectors of the present invention may be constructed usingstandard recombinant techniques widely available to one skilled in theart. Such techniques may be found in common molecular biology referencessuch as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), and PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.).

[0155] Administration of a nucleic acid of the present invention to atarget cell in vivo may be accomplished using any of a variety oftechniques well known to those skilled in the art.

[0156] The vectors of the present invention may be administered orally,parentally, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term parenteral as used hereinincludes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques or intraperitoneally. Suppositories for rectaladministration of the drug can be prepared by mixing the drug with asuitable non-irritating excipient such as cocoa butter and polyethyleneglycols that are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.

[0157] The dosage regimen for treating a disorder or a disease with thevectors of this invention and/or compositions of this invention is basedon a variety of factors, including the type of disease, the age, weight,sex, medical condition of the patient, the severity of the condition,the route of administration, and the particular compound employed. Thus,the dosage regimen may vary widely, but can be determined routinelyusing standard methods.

[0158] The pharmaceutically active compounds (i.e., vectors) of thisinvention can be processed in accordance with conventional methods ofpharmacy to produce medicinal agents for administration to patients,including humans and other mammals. For oral administration, thepharmaceutical composition may be in the form of, for example, acapsule, a tablet, a suspension, or liquid. The pharmaceuticalcomposition is preferably made in the form of a dosage unit containing agiven amount of DNA or viral vector particles (collectively referred toas “vector”). For example, these may contain an amount of vector fromabout 10³-10⁵ viral particles, preferably from about 10⁶-10¹² viralparticles. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, but,once again, can be determined using routine methods. The vector may alsobe administered by injection as a composition with suitable carriersincluding saline, dextrose, or water.

[0159] While the nucleic acids and /or vectors of the invention can beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more vectors of the invention or otheragents. When administered as a combination, the therapeutic agents canbe formulated as separate compositions that are given at the same timeor different times, or the therapeutic agents can be given as a singlecomposition.

[0160] The polypeptide of the present invention may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the protein, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0161] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptide of the present invention may be employed onconjunction with other therapeutic compounds.

[0162] The pharmaceutical compositions may be administered in aconvenient manner, such as the transdermal, transmucosal, enteral andparenteral intravenous routes. The amounts and dosage regimens of TRNAsynthetase polypeptides administered to a subject will depend on anumber of factors, such as the mode of administration, the nature of thecondition being treated, the body weight of the subject being treatedand the judgment of the prescribing physician. Generally speaking, it isgiven, for example, in therapeutically effective doses of at least about10 μg/kg body weight and, in most cases, it would be administered in anamount not in excess of about 8 mg/kg body weight per day and preferablythe dosage is from about 10 μg/kg body weight to about 1 mg/kg bodyweight daily, taking into the account the routes of administration,symptoms, etc.

[0163] The present invention is further directed to inhibiting tRNAsynthetase polypeptides in vivo by the use of antisense technology.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the mature polynucleotide sequence, which encodesfor the polypeptide of the present invention, is used to design anantisense RNA oligonucleotide of from 10 to 40 base pairs in length. ADNA oligonucleotide is designed to be complementary to a region of thegene involved in transcription (triple helix—see Lee et al, Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al, Science, 251: 1360 (1991), thereby preventing transcription andthe production of VEGF2. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of an mRNA molecule into thetRNA synthetase polypeptides (antisense—Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)).

[0164] Alternatively, the oligonucleotides described above can bedelivered to cells by procedures in the art such that the anti-sense RNAor DNA may be expressed in vivo to inhibit production of tRNA synthetasepolypeptides in the manner described above. Antisense constructs to tRNAsynthetase polypeptides, therefore, may inhibit the activity of the tRNAsynthetase polypeptides and prevent the further growth or even regresssolid tumors, since angiogenesis and neovascularization are essentialsteps in solid tumor growth. These antisense constructs may also be usedto treat rheumatoid arthritis, psoriasis and diabetic retinopathy whichare all characterized by abnormal angiogenesis.

[0165] Alternatively, angiostatic trpRS therapy can be used to opposethe oppose the angiogenic activity of endogenous and exogenousangiogenic factors, including tyrRS polypeptides, and to prevent thefurther growth or even regress solid tumors, since angiogenesis andneovascularization are essential steps in solid tumor growth. Suchtherapies can also be used to treat rheumatoid arthritis, psoriasis anddiabetic retinopathy which are all characterized by abnormalangiogenesis.

[0166] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0167] Antibodies generated against the polypeptide corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal, preferably a nonhuman. The antibody so obtained will then bindthe polypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies bindingthe whole native polypeptide. Such antibodies can then be used toisolate the polypeptide from tissue expressing that polypeptide.

[0168] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0169] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.

[0170] Neutralization antibodies can be identified and applied to maskthe activity of tRNA synthetase polypeptides. The utility of such anapproach has been shown in mice model systems against VEGF. VEGF2 canalso be inactivated by certain dominant negative mutants within the geneitself. It is known that both PDGF.alpha. and .beta. form eitherheterodimers or homodimers, and VEGF forms homodimers. These antibodiestherefore may be used to block endogenous angiogenic activity and retardthe growth of solid tumors. These antibodies may also be used to treatinflammation caused by the increased vascular permeability.

[0171] These antibodies may further be used in an immunoassay to detectthe presence of tumors in certain individuals. Enzyme immunoassay can beperformed from the blood sample of an individual.

[0172] The present invention is also directed to antagonist/inhibitorsof the polypeptides of the present invention. The antagonist/inhibitorsare those which inhibit or eliminate the function of the polypeptide.Thus, for example, antagonists bind to a polypeptide of the presentinvention and inhibit or eliminate its function. The antagonist, forexample, could be an antibody against the polypeptide which binds to thepolypeptide or, in some cases, an oligonucleotide. An example of aninhibitor is a small molecule which binds to and occupies the catalyticsite of the polypeptide thereby making the catalytic site inaccessibleto substrate such that normal biological activity is prevented. Examplesof small molecules include but are not limited to small peptides orpeptide-like molecules.

[0173] Alternatively, antagonists to the polypeptides of the presentinvention may be employed which bind to the receptors to which apolypeptide of the present invention normally binds. The antagonists maybe closely related proteins such that they recognize and bind to thereceptor sites of the natural protein, however, they are inactive formsof the natural protein and thereby prevent the action of the normalpolypeptide ligand. The antagonist/inhibitors may be usedtherapeutically as an anti-tumor drug by occupying the receptor sites oftumors. The antagonist/inhibitors may also be used to preventinflammation. The antagonist/inhibitors may also be used to treat solidtumor growth, diabetic retinopathy, psoriasis and rheumatoid arthritis.The antagonist/inhibitors may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinabove described.

[0174] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0175] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene, which method comprises a step of detecting in a biologicalsample obtained from said subject nucleic acid molecules, if any,comprising a nucleotide sequence that is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom SEQ ID NO:9

[0176] The method for diagnosing a pathological condition can comprise astep of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a SEQ ID NO:9.

[0177] Also preferred is a composition of matter comprising isolatednucleic acid molecules wherein the nucleotide sequences of said nucleicacid molecules comprise a panel of at least two nucleotide sequences,wherein at least one sequence in said panel is at least 95% identical toa sequence of at least 50 contiguous nucleotides SEQ ID NO:9. Thenucleic acid molecules can comprise DNA molecules or RNA molecules.

[0178] Further preferred is a method for detecting in a biologicalsample a polypeptide comprising an amino acid sequence which is at least90% identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of amino acid sequences ofSEQ ID NO:9, which method comprises a step of comparing an amino acidsequence of at least one polypeptide molecule in said sample with asequence selected from said group and determining whether the sequenceof said polypeptide molecule in said sample is at least 90% identical tosaid sequence of at least 10 contiguous amino acids of SEQ ID NO:9.

[0179] Also preferred is the above method for identifying the species,tissue or cell type of a biological sample, which method comprises astep of detecting polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from theabove group.

[0180] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene, which method comprises a step of detecting in a biologicalsample obtained from said subject polypeptide molecules comprising anamino acid sequence in a panel of at least two amino acid sequences,wherein at least one sequence in said panel is at least 90% identical toa sequence of at least 10 contiguous amino acids of SEQ ID NO:9

[0181] The present invention also includes a diagnostic system,preferably in kit form, for assaying for the presence of the polypeptideof the present invention in a body sample, such brain tissue, cellsuspensions or tissue sections; or a body fluid sample, such as CSF,blood, plasma or serum, where it is desirable to detect the presence,and preferably the amount, of the polypeptide of this invention in thesample according to the diagnostic methods described herein.

[0182] In a related embodiment, a nucleic acid molecule can be used as aprobe (i.e., an oligonucleotide) to detect the presence of apolynucleotide of the present invention, a gene corresponding to apolynucleotide of the present invention, or a mRNA in a cell that isdiagnostic for the presence or expression of a polypeptide of thepresent invention in the cell. The nucleic acid molecule probes can beof a variety of lengths from at least about 10, suitably about 10 toabout 5000 nucleotides long, although they will typically be about 20 to500 nucleotides in length. Hybridization methods are extremely wellknown in the art and will not be described further here.

[0183] In a related embodiment, detection of genes corresponding to thepolynucleotides of the present invention can be conducted by primerextension reactions such as the polymerase chain reaction (PCR). To thatend, PCR primers are utilized in pairs, as is well known, based on thenucleotide sequence of the gene to be detected. Preferably, thenucleotide sequence is a portion of the nucleotide sequence of apolynucleotide of the present invention. Particularly preferred PCRprimers can be derived from any portion of a DNA sequence encoding apolypeptide of the present invention, but are preferentially fromregions which are not conserved in other cellular proteins.

[0184] Preferred PCR primer pairs useful for detecting the genescorresponding to the polynucleotides of the present invention andexpression of these genes are described in the Examples, including thecorresponding Tables. Nucleotide primers from the corresponding regionof the polypeptides of the present invention described herein arereadily prepared and used as PCR primers for detection of the presenceor expression of the corresponding gene in any of a variety of tissues.

[0185] The present invention also provides a screening assay forantiangiogenic compounds. As disclosed herein, antiangiogenic compounds,such as trpRS polypeptides, can be detected by their ability to opposethe angiogenic effects of tyrRS polypeptides on model systems such aschick allantoic membrane (CAM), and corneal vascularization models.

[0186] The diagnostic system includes, in an amount sufficient toperform at least one assay, a subject polypeptide of the presentinvention, a subject antibody or monoclonal antibody, and/or a subjectnucleic acid molecule probe of the present invention, as a separatelypackaged reagent.

[0187] In another embodiment, a diagnostic system, preferably in kitform, is contemplated for assaying for the presence of the polypeptideof the present invention or an antibody immunoreactive with thepolypeptide of the present invention in a body fluid sample. Suchdiagnostic kit would be useful for monitoring the fate of atherapeutically administered polypeptide of the present invention or anantibody immunoreactive with the polypeptide of the present invention.The system includes, in an amount sufficient for at least one assay, apolypeptide of the present invention and/or a subject antibody as aseparately packaged immunochemical reagent.

[0188] Instructions for use of the packaged reagent(s) are alsotypically included.

[0189] A diagnostic system of the present invention preferably alsoincludes a label or indicating means capable of signaling the formationof an immunocomplex containing a polypeptide or antibody molecule of thepresent invention.

[0190] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturing them toform a fluorochrome (dye) that is a useful immunofluorescent tracer.Suitable fluorescent labeling agents are fluorochromes such asfluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference. Other suitable labeling agents areknown to those skilled in the art.

[0191] In preferred embodiments, the indicating group is an enzyme, suchas horseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

[0192] Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radio labeling agent is aradioactive element that produces gamma ray emissions. Elements whichthemselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Crrepresent one class of gamma ray emission-producing radioactive elementindicating groups. Particularly preferred is ¹²⁵I. Another group ofuseful labeling means are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³Nwhich themselves emit positrons. The positrons so emitted produce gammarays upon encounters with electrons present in the animal's body. Alsouseful is a beta emitter, such ¹¹¹indium or ³H.

[0193] The linking of labels or labeling of polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium (see, e.g., Galfre et al., Meth. Enzymol., 73:3-46(1981)). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable (see, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Pat. No.4,493,795).

[0194] The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. Preferably the specific binding agentbinds the reagent species when that species is present as part of acomplex.

[0195] In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

[0196] The diagnostic kits of the present invention can be used in an“ELISA” format to detect the quantity of the polypeptide of the presentinvention in a sample.

[0197] Thus, in some embodiments, a polypeptide of the presentinvention, an antibody or a monoclonal antibody of the present inventioncan be affixed to a solid matrix to form a solid support that comprisesa package in the subject diagnostic systems.

[0198] A reagent is typically affixed to a solid matrix by adsorptionfrom an aqueous medium, although other modes of affixation applicable toproteins and polypeptides can be used that are well known to thoseskilled in the art. Exemplary adsorption methods are described herein.

[0199] Useful solid matrices are also well known in the art. Suchmaterials are water insoluble and include the cross-linked dextranavailable under the trademark SEPHADEX from Pharmacia Fine Chemicals(Piscataway, N.J.), agarose; polystyrene beads of ut 1 micron (μm) toabout 5 millimeters (mm) in diameter available from several suppliers(e.g., Abbott Laboratories, Chicago, Ill.), polyvinyl chloride,polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-basedwebs (sheets, strips or paddles) or tubes, plates or the wells of amicro titer plate, such as those made from polystyrene orpolyvinylchloride.

[0200] The reagent species, labeled specific binding agent, oramplifying reagent of any diagnostic system described herein can beprovided in solution, as a liquid dispersion or as a substantially drypower, e.g., in lyophilized form. Where the indicating means is anenzyme, the enzyme's substrate can also be provided in a separatepackage of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separatelypackaged elements in this diagnostic assay system.

[0201] The packaging materials discussed herein in relation todiagnostic systems are those customarily utilized in diagnostic systems.

[0202] The preferred embodiment of the present invention is bestunderstood by referring to the FIGURES and the Examples below. TheExamples, which follow, are illustrative of specific embodiments of theinvention, and various uses thereof. They are set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

EXAMPLE 1 Preparation of Endotoxin-Free Recombinant TyrRS and TrpRS

[0203] Endotoxin-free recombinant human TyrRS and human TrpRS wasprepared as follows. Plasmids encoding full-length TyrRS (528 amino acidresidues; FIGS. 1 and 15), truncated TyrRS (mini TyrRS, residues 1-364of full-length TyrRS; carboxyl-domain of TyrRS, residues 359-528 offull-length TyrRS), full-length TrpRS (471 amino acid residues), ortruncated TrpRS (mini TrpRS, residues 48-471 of full-length TrpRS;supermini TrpRS, residues 71-471 of full-length TrpRS), each alsoencoding a C-terminal tag of six histidine residues, were introducedinto E. coli strain BL 21 (DE 3) (Novagen, Madison, Wis.). Human matureEMAPII, also encoding a C-terminal tag of six histidine residues, wassimilarly prepared for use in some Examples. Over expression ofrecombinant TyrRS or TrpRS was induced by treating the cells withisopropyl β-D-thiogalactopyranoside for 4 hours. Cells were then lysedand the proteins from the supernatant purified on His•Bind®nickelaffinity columns (Novagen) according to the manufacturer's suggestedprotocol. Following purification, TrpRS proteins were incubated withphosphate-buffered saline (PBS) containing 1 μM ZnSO₄ and then free Zn²⁺was removed (Kisselev et al., 1981, Eur. J. Biochem. 120:511-17).

[0204] Endotoxin was removed from protein samples by phase separationusing Triton X-114 (Liu et al., 1997, Clin. Biochem. 30:455-63). Proteinsamples were determined to contain less than 0.01 units of endotoxin permL using an E-Toxate® gel-clot assay (Sigma, St. Louis, Mo.). Proteinconcentration was determined by the Bradford assay (Bio-Rad, Hercules,Calif.) using bovine serum albumin (BSA) as a standard.

EXAMPLE 2 Cytokine Activity of Human TyrRS

[0205] Human full-length TyrRS (SEQ ID NO:1), human mini TyrRS (SEQ IDNO:3), human TyrRS carboxyl-terminal domain (SEQ ID NO:5), human EMAPII, and E. coli TyrRS were analyzed for cytokine activity in assaysexamining MP or PMN chemotaxis, MP production of TNFα or tissue factor,or PMN release of myeloperoxidase.

[0206] Cells for the various cytokine assays described were preparedfrom acid citrate dextrose-treated blood of normal healthy volunteers.Human PMNs were isolated from the blood by centrifugation (700 xg) overHistopaque 1077 and 1119 (Sigma). Fractions containing PMNs were exposedto 0.2% NaCl for 30 seconds to lyse erythrocytes, immediately restoredto isotonicity by the addition of 1.6% NaCl, and then centrifuged for 10minutes. This procedure was repeated twice. Human MPs were isolated bycentrifugation on Histopaque 1077 (Sigma). The mononuclear fraction wasobtained, washed twice in Hanks' balanced salt solution, resuspended inRPMI-1640 medium (Sigma) containing 10% heat-inactivated fetal bovineserum (Sigma), plated in tissue culture flasks, and incubated in a 6%CO₂ incubator at 37° C. for 1-2 hours (Kumagai et al., 1979, J. Immunol.Methods 29:17-25). Nonadherent cells were removed by washing the flasksthree times with Hanks' balanced salt solution, and adherent cells wereharvested by incubation with calcium-magnesium free phosphate-bufferedsaline containing 2 mM EDTA for 15 minutes at 4° C., followed byextensive washing.

[0207] MP chemotaxis assays were performed in a ChemoTX microchemotaxischamber (Neuro Probe, Gaithersburg, Md.) containing polycarbonatefilters (5 μm pores) with polyvinylpyrrolidone (PVP). MPs were suspendedin RPMI-1640 medium containing 1% heat-inactivated fetal bovine serum,and 10⁴ cells were added to the upper chamber. Sample proteins (1 nM)were added to the lower compartment of chemotaxis chambers, and thechambers were incubated for 3 hours. After incubation, nonmigratingcells were removed, membranes were fixed in methanol, and migratingcells were visualized with the Hemacolor™ stain set (EM DiagnosticSystems, Gibbstown, N.J.). Migrating cells were counted in high-powerfields (HPFs). Each determination shown in FIG. 2 represents the averageof nine HPF measurements.

[0208] MP TNFα production was examined following incubation of 10⁵ MPswith 1 nM of sample protein for 14 hours. Aliquots of the culturesupernatant were then assayed fpr TNFα production using a TNFαenzyme-linked immunosorbent assay kit (Sigma). Each determination shownin FIG. 2 represents the mean of four measurements from at least threeindependent experiments.

[0209] MP tissue factor production was examined following incubation of10⁴ MPs with 1 nM of sample protein for 4 hours. Tissue factor activitywas then inferred from measurements of Factor Vlla-dependent Factor Xaformation (Wolfson et al., 1990 J. Chromatogr. 503:277-81). Eachdetermination shown in FIG. 2 represents the mean of four measurementsfrom at least three independent experiments.

[0210] PMN release of myeloperoxidase was examined following incubationof 3×10⁶ PMNs per mL with 1 nM of sample protein for 60 minutes. Thegeneration of peroxidase was then measured by the reduction of3,3′,5,5′-tetramethylbenzidine (Barker et al., 1982, FEBS Lett.150:419-23). Peroxidase activity is shown in FIG. 3 as the percent oftotal peroxidase activity where 100% peroxidase activity is defined asthe activity observed for 3×10⁶ PMNs following exposure to 10 μM phorbolester for 60 minutes. Each determination shown in FIG. 3 represents themean of four measurements from at least three independent experiments.

[0211] PMN chemotaxis assays were performed in a ChemoTX microchemotaxischamber (described herein). Sample proteins (1 nM) were added to thelower compartment of chemotaxis chambers, and 10⁴ PMNs were added to theupper compartment. Chambers were incubated for 45 min, and migratingcells were counted in HPFs. Each determination shown in FIG. 2represents the average of nine HPF measurements.

[0212] As shown in FIG. 2 (white bars), TyrRS carboxyl-terminal domaininduced MP migration to an extent comparable with that observed for EMAPII. In contrast, no chemotaxis was observed with full-length TyrRS. TheTyrRS carboxyl-terminal domain also stimulated production of TNFα (FIG.2, gray bars) and tissue factor (FIG. 3, white bars) in MPs, induced therelease of myeloperoxidase in PMNs (FIG. 3, gray bars), and induced PMNmigration (FIG. 4). The induction of PMN migration by the TyrRScarboxyl-terminal domain and EMAP II showed the bell-shapedconcentration dependence that is characteristic of chemotactic cytokines(Wakasugi et al., supra). Full-length TyrRS had none of the propertiesobserved for the carboxyl-terminal domain (FIGS. 2-4).

[0213] The cytokine activity of the amino-terminal catalytic domain ofTyrRS (mini TyrRS) examined in parallel with that of the TyrRScarboxyl-terminal domain. Mini TyrRS did not induce MP migration (FIG.2, white bars) and did not stimulate production of TNFA (FIG. 2, graybars) or tissue factor (FIG. 3, white bars) in MPs. Surprisingly, miniTyrRS did induce PMN migration (FIG. 4), and this activity showed abell-shaped concentration dependence. These results suggest that miniTyrRS is a leukocyte chemoattractant. The PMN response to mini TyrRSadded to the lower compartment of a chemotaxis chamber was attenuated bythe addition of mini TyrRS to the upper well, indicating that enhancedPMN migration was due to chemotaxis, not simply chemokinesis (stimulatedrandom movement). E. coli TyrRS, which is similar in size to human miniTyrRS, was inactive in all of the assays (FIGS. 2-4).

EXAMPLE 3 Secretion of Human TyrRS From U-937 Cells

[0214] In order to examine whether human TyrRS, like human EMAP II, issecreted from apoptotic tumor cells, human histiocytic lymphoma U-937cells were first grown in serum-free medium to induce apoptosis. Priorto growth in serum-free medium, U-937 cells were maintained in RPMI-1640medium containing 10% heat-treated FBS (Sigma), 100 U/mL penicillin and100 μg/mL streptomycin (Sigma) in an atmosphere of 6% CO₂ in air at 37°C. U-937 cells were maintained in logarithmic growth phase by routinepassage every 2-3 days. For serum-free growth, 4×10⁶ U-937 cells werecultured in RPMI-1640 medium without FBS for 24 hours. Apoptosis ofU-937 cells was verified by DNA fragment assay, in which thecharacteristic DNA ladder for apoptotic cells was observed on an agarosegel.

[0215] Cell supernatants were collected following growth of U-937 cellsin serum-free media for 4, 12, or 24 hours. These supernatants were thenexamined by Western blot analysis using a rabbit polyclonal anti-TyrRSantibody. To examine proteins in the cell supernatant, 20 mL of spentculture medium was first treated with 2 mM PMSF, 10 μg/mL aprotinin, 20μg/mL leupeptin, and 10 μg/mL pepstatin A. Treated culture medium wasconcentrated using Centriprep-10 columns (Amicon, Beverly, Mass.) andthen separated on a 12.5% SDS-polyacrylamide gel. Following transferonto an Immobilon-P™ membrane (Millipore, Bedford, Mass.), blots wereblocked with PBS and 3% BSA and incubated with rabbit polyclonalanti-TyrRS antibodies. After washing, blots were incubated with a 1:4000dilution of horseradish peroxidase-linked anti-rabbit IgG (Amersham LifeScience, Arlington Heights, Ill.) for detection of TyrRS.

[0216] Cell lysates were prepared by first washing collected U-937 cellstwice with ice-cold PBS and then resuspending the cells in lysis buffercontaining 25 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM dithiothreitol, 0.1%CHAPS, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/mL aprotinin, 20μg/mL leupeptin, and 10 μg/mL pepstatin A. Cells were then frozen andthawed three times in liquid nitrogen and centrifuged for 30 minutes at4° C.

[0217] Protein immunoblot analysis of the cell supernatant fraction,with a polyclonal antibody to human TyrRS, revealed that full-lengthTyrRS was secreted from apoptotic tumor cells, but not from cells undernormal conditions (FIG. 7A). Under apoptotic conditions, the amount ofsecreted human full-length TyrRS increased with the incubation time(FIG. 7B). After 24 hours of growth in serum-free medium, more than 50%of the total native TyrRS was released from the cells. A similarproportion of mature EMAP II is secreted from U-937 cells under the sameconditions (Kao, et al., 1994, J. Biol. Chem. 269:25106-19).

[0218] To exclude the possibility that the apparent secretion of TyrRSwas due to cell lysis, the activity of cytosolic lactate dehydrogenase(LDH) in the supernatants was measured. LDH activities were determinedspectrophotometrically with a CytoTox 96 Non-Radioactive CytotoxicityAssay kit (Promega, Madison, Wis.). LDH activity in the supernatants wasless than 10% of that in cell extracts and did not increase even after72 hours of incubation. These results are consistent with the hypothesisthat the increase of TyrRS in the supernatants is due to proteinsecretion.

[0219] The permeability of the “secreting” apoptotic cells was alsoexamined using Trypan Blue exclusion as a test for intact cells. Theapoptotic cells did not take up the stain, indicating that cell lysiswas not responsible for the appearance of TyrRS in the apoptotic cellsupernatant. As a further control, human alanyl-tRNA synthetase(AlaRS),which possesses none of the cytokine-like motifs of human TyrRS, wasexamined for possible secretion. Protein immunoblot analysis showedthat, under the same apoptoticconditions, no AlaRS was secreted. Theactivities of four other aminoacyl-tRNA synthetases in the supernatantsor cell extracts of apoptotic U-937 cells were also studied. When cellextracts were used in assays with bovine tRNA, aminoacylation wasobserved only for alanine, isoleucine, lysine, valine, and tyrosine. Incontrast, when supernatants were used, only tyrosine was aminoacylated.

EXAMPLE 4 Cleavage of Human TyrRS by PMN Elastase

[0220] In order to examine whether full-length TyrRS can be cleaved byPMN elastase, a protease released from PMNs (Wright, et al., 1992, J.Cell. Biochem. 48:344-55), full-length TyrRS was treated with PMNelastase in PBS (pH 7.4) at a protease:protein ratio of 1:3000 for 30minutes at 37° C. Following cleavage, the sample was separated on a12.5% SDS-polyacrylamide gel along with untreated human full-lengthTyrRS, human mini TyrRS, the human TyrRS carboxyl-terminal domain, and ahuman extended TyrRS carboxyl-terminal domain. Immunoblot analysis wasperformed as described in Example 3.

[0221] As shown in FIG. 8A, the addition of PMN elastase to full-lengthTyrRS generated a doublet of ˜40 kD fragments and a ˜24 kD fragment. The˜40 kD fragments have a molecular weight similar to that of mini TyrRS.Sequence analysis of the ˜40 kDfragments revealed that each has anamino-terminal sequence of M-G-D-A-P(SEQ ID NO:56), as does humanfull-length TyrRS. FIG. 8B illustrates the local sequence comparisonbetween human pro-EMAP II and human full-length TyrRS in the regionsnear their cleavage sites.

[0222] Immunoblot analysis further revealed that the ˜24 kD fragment isa carboxyl-terminal domain. A recombinant extended TyrRScarobxyl-terminal domain (residues 344-528 of human full-length TyrRS)was prepared to more closely reproduce the putative cleavagesiterecognized by PMN elastase. Both the extended TyrRS carboxyl-terminaldomain and the PMN elastase ˜24 kD cleavage product migrated to similarpositions on an SDS-polyacrylamide gel (FIG. 8A). Subsequent experimentsdemonstrated that the extended carboxyl-terminal domain was capable ofcan inducing both MP and PMN chemotaxis. In experiments examining theability of a recombinant truncated mini TyrRS (comprising residues 1-344of human full-length TyrRS in comparison to mini TyrRS which comprisesTyrRS residues 1-364) to act as a chemoattractant, the truncated miniTyrRS was capable of functioning as a chemoattractant for PMNs but notfor MPs.

[0223] In vivo cleavage analysis was performed in IL-8-stimulated PMNs,which release PMN elastase (Bjørnland et al., 1998, Int. J. Oncol.12:535-40). Recombinant human full-length TyrRS. when added to suchcells, was cleaved into ˜40 kD and ˜24 kD fragments. When full-lengthTyrRS was added to nonstimulated PMNs, no TyrRS cleavage was observed.Immunoblot analysis, using antibodies specific for the amino-terminaland carboxyl-terminal domains indicated that the ˜40 kD and ˜24 kDfragments comprised mini TyrRS and the TyrRS carboxyl-terminal domain,respectively. When native TyrRS, which was isolated from apoptotic U-937cells, was added to IL-8-stimulated PMNs, the same ˜40 kD and ˜24 kDfragments were generated.

[0224] The demonstration that human full-length TyrRS can be split intotwo distinct cytokines, suggests that there is a link between proteinsynthesis and signal transduction. In principle, the secretion of anessential component of the translational apparatus as an early event inapoptosis would be expected to arrest translation and thereby accelerateapoptosis. The secreted TyrRS cytokines could function as intercellularsignal transducers, attracting PMNs and thus amplifying the localconcentration of PMN elastase. This recursive cycle could enhancecleavage of secreted human TyrRS, thereby enhancing recruitment ofmacrophages to sites of apoptosis, which would promote removal of cellcorpses.

EXAMPLE 5 Induction of Endothelial Cell Migration by Human TyrRS

[0225] All α-chemokines containing the ELR motif, such as IL-8, act asangiogenic factors (Strieter et al., 1995, J. Biol. Chem. 270:27348-57).In contrast, α-chemokines lacking the ELR motif act as angiostaticfactors (id.). In order to evaluate the angiogenic activity of TyrRS,which contains the ELR motif, human full-length TyrRS and human miniTyrRS were first examined for their ability to induce endothelial cellmigration. Such angiogenic assays were performed using human umbilicalvein endothelial cells (HUVECs) (Clonetics, Walkersville, Md.). Cellswere maintained in EGM-2® BulletKit® medium (Clonetics) in an atmosphereof 6% CO₂ at 37° C. according to the supplier's instructions.

[0226] Cell migration assays were performed using the modified Boydenchamber (6.5 mm Transwells) with polycarbonate membranes (8.0 μm poresize) (Costar Corp., Cambridge, Mass.) (Masood et al., 1999, Blood93:1038-44). Wells were coated overnight with 25 μg/mL human fibronectin(Biosource International, Camarillo, Calif.) in PBS and then allowed toair-dry. HUVECs were suspended in Dulbecco's modified Eagle's medium(DMEM) (Gibco-BRL, Gaithersburg, Md.) containing 0.1% BSA (Sigma) and2×10⁵ cells per well were added to the upper chamber. The chemotacticstimulus (50 nM of a given TyrRS molecule or 0.5 nM of human vascularendothelial growth factor-165 (VEGF₁₆₅) (Biosource International,Camarillo, Calif.)) was placed in the lower chamber, and the cells wereallowed to migrate for 6 hours at 37° C. in a 6% CO₂ incubator. Afterincubation, non-migrant cells were removed from the upper face of theTranswell membrane with a cotton swab and migrant cells, those attachedto the lower face, were fixed in methanol and visualized with theHemacolor® stain set (EM Diagnostic Systems, Gibbstown, N.J.). Migratingcells were counted in high-power fields (HPFs). HUVECs were suspended inmedia with the inhibitor for 30 minutes before placement in the chamber.Each determination shown in FIG. 16 represents the average of nine HPFmeasurements.

[0227] As shown in FIG. 16, human mini TyrRS stimulated induction ofHUVEC chemotaxis as did the positive control VEGF₁₆₅. In contrast, nochemotaxis was observed with human full-length TyrRS or human mini TyrRSmutant (containing ELQ in place of conserved ELR motif). The ability ofmini TyrRS to induce directed migration of endothelial cells supportedthe notion that mini TyrRS also may induce angiogenesis in vivo.

EXAMPLE 6 Induction of in vivo Angiogenesis by Human TyrRS

[0228] In vivo angiogenesis assays were conducted in chickchorioallantoic membrane (CAM) (Nicolaou et al., 1998, Bioorg. Med.Chem. 6:1185-208). Ten-day-old chick embryos were purchased fromMcintyre Poultry (Lakeside, Calif.) and were incubated at 37° C. and 70%humidity. A small hole was made with a small crafts drill (Dremel,Emerson Electric, Racine, Wis.) directly over the air sac at the end ofthe egg. The embryos were candied to determine a location to drill asecond hole directly over embryonic blood vessels. Negative pressure wasapplied to the original hole, which resulted in the chorioallantoicmembrane (CAM) pulling away from the shell membrane and creating a falseair sac. A window was cut in the eggshell over the dropped CAM, exposingthe CAM to direct access for experimental manipulation. Cortisoneacetate-treated 5 mm filter disks were soaked with a particular proteinsample (25 ng of VEGF₁₆₅ or 250 ng of a given TyrRS molecule) and thefilter disks added directly to the CAMs, which were relatively devoid ofpreexisting blood vessels. The windows were sealed with sterile tape andincubated at 37° C. At 0, 24, and 48 hours following incubation, 3 μg ofinterferon-alpha inducible protein (IP-10) (R & D Systems, Minneapolis,Minn.) was topically applied to the filter disks. After 72 hours, theCAM tissue associated with the filter disk was harvested and quantifiedusing a stereo microscope. Angiogenesis was assessed as the number ofvisible blood vessel branch points within the defined area of the filterdisks. Each determination shown in FIG. 17 represents the mean from 5-8embryos.

[0229] As shown in FIG. 17, human mini TyrRS induced angiogenesis as didthe positive control human VEGF₁₆₅. Moreover, the angiogenesisstimulated by both mini TyrRS and VEGF₁₆₅ was inhibited by theangiostatic α-chemokine, IP-10. Human mini TyrRS mutant failed to inducein vivo angiogenesis, suggesting that the ELR motif of mini TyrRS is asimportant for angiogenesis as the motif is for the angiogenic activityof α-chemokines.

EXAMPLE 7 Angiostatic Effect of Human TrpRS on Cell Proliferation

[0230] Expression of mini TrpRS in human cells is highly stimulated bythe addition of interferon-γ (IFN-γ) (Shaw et al., 1999, Electrophoresis20:984-93). Expression of the α-chemokines IP-10 (interferon-γ inducibleprotein) and MIG (monokine induced by interferon-γ) has also been shownto be enhanced by IFN-γ (Kaplan et al., 1987, J. Exp. Med. 166:1098-108; Farber, 1993, Biochem. Biophys. Res. Commun. 192:223-30). Theseα-chemokines lack the ELR motif and function as angiostatic factors bothin vitro and in vivo (Strieter et al., supra). The presence in mammalianTrpRS molecules of a Rossmann nucleotide binding fold and DLT sequence,in place of the ELR motif, suggests that mammalian TrpRS molecules mayfunction as angiostatic factors.

[0231] The angiostatic activity of TrpRS was first evaluated inexperiments testing the ability of human full-length TrpRS and humanmini TrpRS to inhibit human VEGF₁₆₅-induced cell proliferation. Tissueculture-treated 96-well plates (Coming Costar Corp., Cambridge, Mass.)were coated with 0.1% gelatin (Sigma) overnight. Cells were then seededin the gelatinized plates at a density of 5×10³ cells per well in DMEMmedium (Gibco-BRL) containing heat-inactivated fetal bovine serum (FBS)(10%, Sigma) and penicillin/streptomycin (100 units/mL-100 μg/mL,Sigma). The following day, the cells were treated with 2 μM of a givenTrpRS in the presence of 2 nM VEGF₁₆₅. After 72 hours of incubation,assays were performed by using the CellTiter 96® aqueous one-solutioncell proliferation assay kit (Promega, Madison, Wis.). Results of theinhibition assay are shown in FIG. 18 as the percentage of netproliferation of VEGF₁₆₅. Each determination shown represents the meanof five experiments.

[0232] As shown in FIG. 18, human full-length TrpRS exhibited noangiostatic activity and human mini TrpRS was able to inhibit humanVEGF₁₆₅-induced cell proliferation.

EXAMPLE 8 Angiostatic Effect of Human TrpRS on Endothelial CellMigration

[0233] The angiostatic activity of TrpRS was next evaluated inexperiments testing the ability of human full-length TrpRS and humanmini TrpRS to inhibit human VEGF₁₆₅-induced or human mini TyrRS-inducedcell migration. Cell migration assays were performed as described inExample 5 with full- length TrpRS or mini TrpRS added to VEGF₁₆₅-inducedor mini TyrRS-induced HUVEC samples. HUVEC samples were treated with 0.5nM VEGF₁₆₅, 50 nM mini TyrRS, and 500 nM of a given TrpRS. Fourmeasurements of chemotaxis were done for each protein. Eachdetermination shown in FIG. 19 represents the average of nine HPFmeasurements.

[0234] As shown in FIG. 19, human mini TrpRS inhibited humanVEGF₁₆₅-induced and human mini TyrRS-induced HUVEC chemotaxis. Incontrast, human full-length TrpRS had no effect on VEGF₁₆₅-induced ormini TyrRS-induced HUVEC chemotaxis.

EXAMPLE 9 Angiostatic Effect of Human TrpRS on in vivo Angiogenesis

[0235] The angiostatic activity of human full-length TrpRS and humanmini TrpRS was also analyzed in in vivo angiogenesis assays conducted inchick CAM. In vivo angiogenesis assays were performed as described inExample 6 with 3 μg of full-length TrpRS or mini TrpRS added toVEGF₁₆₅-induced or mini TyrRS-induced CAM tissue.

[0236] As shown in FIG. 20, the angiogenic activity of human VEGF₁₆₅ andhuman mini TyrRS was inhibited by human mini TrpRS. Human full-lengthTrpRS had no observable angiostatic activity.

EXAMPLE 10 Secretion of Human TrpRS From U-937 Cells

[0237] As shown in Example 3, TyrRS is secreted from apoptotic tumorcells where it can be cleaved by PMN elastase to release mini TyrRS andan EMAP II-like carboxyl-domain. In contrast, several other tRNAsynthetases, including AlaRS, LysRS, IleRS, and VaIRS, were not found tobe secreted under the same conditions.

[0238] In order to determine whether TrpRS could be secreted under theseconditions, secretion assays using U-937 cells were performed asdescribed in Example 3. Cell supernatants were examined by Western blotanalysis using a polyclonal anti-TrpRS antibody.

[0239] As shown in FIG. 21, human full-length TrpRS was secreted fromapoptotic U-937 cells, but not from U-937 cells maintained under normal(i.e., serum) conditions. It was not possible to determine whether humanmini TrpRS was also secreted from apoptotic tumor cells, since theamount of mini TrpRS generated from full-length TrpRS is comparativelysmall.

EXAMPLE 11 Cleavage of Human TrpRS by PMN Elastase

[0240] Cleavage of human full-length TrpRS by PMN elastase was examinedusing the procedures similar to those described in Example 4. TrpRS wastreated with PMN elastase in PBS (pH 7.4) at a protease:protein ratio of1:3000 for 0, 15, 30, or 60 minutes. Following cleavage, samples wereanalyzed on 12.5% SDS-polyacrylamide gels. As shown in FIG. 22, PMNelastase cleavage of the 54 kDa full-length TrpRS generated a majorfragment of 47 kDa and a minor fragment of 44 kDa. Both fragments weresimilar in size to the 49 kDa mini TrpRS fragment.

[0241] Western blot analysis with antibodies directed against thecarboxyl-terminal His₆-tag of the recombinant TrpRS protein revealedthat both fragments possessed the His₆-tag at their carboxyl-terninus.Thus, only the amino-terminus of two TrpRS fragments has been truncated.The amino-terminal sequences of the TrpRS fragments were determined byEdman degradation using an ABI Model 494 sequencer. Sequencing of thesefragments showed that the amino-terminal sequences were S-N-H-G-P (SEQID NO:57) and S-A-K-G-I (SEQ ID NO:58), indicating that theamino-terminal residues of the major and minor TrpRS fragments werelocated at positions 71 and 94, respectively, of full-length TrpRS.

[0242] The angiostatic activity of the major and minor TrpRS fragmentswas analyzed in HUVEC proliferation, HUVEC migration, and chick CAM invivo angiogenesis assays (as described in Examples 7-9). Recombinantforms of the major and minor TrpRS fragments were prepared for use inthese assays. As shown in FIG. 23, the major TrpRS fragment (designatedas supermini TrpRS) was capable of inhibiting VEGF₁₆₅-stimulated orhuman mini TyrRS-stimulated angiogenesis. Supermini TrpRS was alsocapable of inhibiting HUVEC proliferation and migration. Thus, the majorproduct of PMN elastase digestion of full-length TrpRS has angiostaticactivity. In the aforementioned assays angiostatic activity was notnoted for the minor Trp-RS fragment, but angiostatic activity wasindicated in a post-natal mouse retinal angiogenesis model.

EXAMPLE 12 Human Mini TrpRS Primary Structure

[0243] Angiogenic mini TyrRS and angiostatic mini TrpRS and superminiTrpRS have their respective ELR and DLT motifs imbedded in closelysimilar Rossmann binding fold domains. In previous experiments,α-chemokines possessing the ELR motif have been shown to act asangiogenic factors and α-chemokines lacking the ELR motif have beenshown to act as angiostatic factors (Strieter et al., supra). It appearsthat the biological balance of angiogenic (ELR) and angiostatic(non-ELR) α-chemokines regulates angiogenesis (id.). For example, theanti-tumor genic IFN-γ induces production of IP-10 and MIG (angiostaticα-chemokines) and also attenuates the expression of IL-8 (angiogenicα-chemokine) (Gusella et al., 1993, J. Immunol. 151:2725-32). Similarly,in some cell lines TrpRS is highly expressed in the presence of IFN-γ

[0244] Mammalian TrpRS shares some sequence similarity with semaphorin-Eand neuropilin-2 (FIGS. 24 and 25). Neuropilin is a receptor for twounrelated ligands with disparate activities: VEGF₁₆₅, an angiogenicfactor, and semaphorins, which are mediators of neuronal guidance (Chenet al., 1998, Neuron 21:1273-82; Neufeld et al., 1999, FASEB J.13:9-22). Semaphorin-E is also a ligand for neuropilin-2 and semaphorinhas been shown to be capable of blocking the binding of VEGF₁₆₅ toneuropilin (Chen et al., supra). The sequence similarity of a portion ofsemaphorin-E to TrpRS suggests that human mini TrpRS and human superminiTrpRS bind neuropilin-2, thereby inhibiting VEGF₁₆₅ binding toneuropilin-2. Since the shared sequence similarity between neuropilin-2and TrpRS is located in the neuropilin-2 c- domain that is required forneuropilin-2 dimerization (Chen et al., supra) (FIG. 25), human miniTrpRS and human supermini TrpRS may inhibit the dimerization ofneuropilin-2.

[0245] Mature EMAP II has been reported to be a cytokine withanti-angiogenic properties (Schwarz et al., 1999, J. Exp. Med.190:341-54). The carboxyl-domain of human TyrRS has also been shown tohave an EMAP II-like cytokine activity (see Example 2). Angiostatic miniTrpRS may be produced by alternative splicing of the pre-mRNA or, asshown in Example 11, angiostatic supermini TrpRS can be generated by PMNelastase cleavage. PMN elastase has been shown to be present in humancolorectal carcinoma with particular enrichment at the tumor-hostinterface (Bjørnland et al., supra). Also, breast and non-small celllung cancer cells are known to secrete PMN elastase in vitro (Yamashitaet al., 1996, Chest 109:1328-34). Thus, human TyrRS and TrpRS, which aresecreted from apoptotic cells, may be cleaved by PMN elastase at thetumor-host interface. The cleaved enzymes can then act as angiogenic andangiostatic factors that regulate tumor invasion.

[0246] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

1 58 1 5174 DNA Artificial Sequence CDS (3428)..(5035) Description ofArtificial Sequence human full-length TyrRS in pET20B 1 tggcgaatgggacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgaccgctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgccacgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgatttagtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtgggccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagtggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgatttataagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaatttaacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaatgtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatgagacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaacatttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcacccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttacatcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgttttccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgccgggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactcaccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgccataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaaggagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaaccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatggcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaattaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccggctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcattgcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagtcaggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaagcattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcatttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatcccttaacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttcttgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctaccagcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttcagcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttcaagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgctgccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataaggcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacctacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaagggagaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggagcttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgacttgagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaacgcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcgttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgccgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatgcggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctcagtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtgactgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttgtctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtcagaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtggtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctccagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctgtttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgatgaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactggaacgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactcagggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagcatcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagactttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgcagcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggcaaccccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggccaggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggagaccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atgggg gac gct ccc agc cct gaa gag aaa ctg cac ctt atc 3469 Met Gly Asp AlaPro Ser Pro Glu Glu Lys Leu His Leu Ile 1 5 10 acc cgg aac ctg cag gaggtt ctg ggg gaa gag aag ctg aag gag ata 3517 Thr Arg Asn Leu Gln Glu ValLeu Gly Glu Glu Lys Leu Lys Glu Ile 15 20 25 30 ctg aag gag cgg gaa cttaaa att tac tgg gga acg gca acc acg ggc 3565 Leu Lys Glu Arg Glu Leu LysIle Tyr Trp Gly Thr Ala Thr Thr Gly 35 40 45 aaa cca cat gtg gct tac tttgtg ccc atg tca aag att gca gac ttc 3613 Lys Pro His Val Ala Tyr Phe ValPro Met Ser Lys Ile Ala Asp Phe 50 55 60 tta aag gca ggg tgt gag gta acaatt ctg ttt gcg gac ctc cac gca 3661 Leu Lys Ala Gly Cys Glu Val Thr IleLeu Phe Ala Asp Leu His Ala 65 70 75 tac ctg gat aac atg aaa gcc cca tgggaa ctt cta gaa ctc cga gtc 3709 Tyr Leu Asp Asn Met Lys Ala Pro Trp GluLeu Leu Glu Leu Arg Val 80 85 90 agt tac tat gag aat gtg atc aaa gca atgctg gag agc att ggt gtg 3757 Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met LeuGlu Ser Ile Gly Val 95 100 105 110 ccc ttg gag aag ctc aag ttc atc aaaggc act gat tac cag ctc agc 3805 Pro Leu Glu Lys Leu Lys Phe Ile Lys GlyThr Asp Tyr Gln Leu Ser 115 120 125 aaa gag tac aca cta gat gtg tac agactc tcc tcc gtg gtc aca cag 3853 Lys Glu Tyr Thr Leu Asp Val Tyr Arg LeuSer Ser Val Val Thr Gln 130 135 140 cac gat tcc aag aag gct gga gct gaggtg gta aag cag gtg gag cac 3901 His Asp Ser Lys Lys Ala Gly Ala Glu ValVal Lys Gln Val Glu His 145 150 155 cct ttg ctg agt ggc ctc tta tac cccgga ctg cag gct ttg gat gaa 3949 Pro Leu Leu Ser Gly Leu Leu Tyr Pro GlyLeu Gln Ala Leu Asp Glu 160 165 170 gag tat tta aaa gta gat gcc caa tttgga ggc att gat cag aga aag 3997 Glu Tyr Leu Lys Val Asp Ala Gln Phe GlyGly Ile Asp Gln Arg Lys 175 180 185 190 att ttc acc ttt gca gag aag tacctc cct gca ctt ggc tat tca aaa 4045 Ile Phe Thr Phe Ala Glu Lys Tyr LeuPro Ala Leu Gly Tyr Ser Lys 195 200 205 cgg gtc cat ctg atg aat cct atggtt cca gga tta aca ggc agc aaa 4093 Arg Val His Leu Met Asn Pro Met ValPro Gly Leu Thr Gly Ser Lys 210 215 220 atg agc tct tca gaa gag gag tccaag att gat ctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu Glu Glu Ser LysIle Asp Leu Leu Asp Arg Lys 225 230 235 gag gat gtg aag aaa aaa ctg aagaag gcc ttc tgt gag cca gga aat 4189 Glu Asp Val Lys Lys Lys Leu Lys LysAla Phe Cys Glu Pro Gly Asn 240 245 250 gtg gag aac aat ggg gtt ctg tccttc atc aag cat gtc ctt ttt ccc 4237 Val Glu Asn Asn Gly Val Leu Ser PheIle Lys His Val Leu Phe Pro 255 260 265 270 ctt aag tcc gag ttt gtg atccta cga gat gag aaa tgg ggt gga aac 4285 Leu Lys Ser Glu Phe Val Ile LeuArg Asp Glu Lys Trp Gly Gly Asn 275 280 285 aaa acc tac aca gct tac gtggac ctg gaa aag gac ttt gct gct gag 4333 Lys Thr Tyr Thr Ala Tyr Val AspLeu Glu Lys Asp Phe Ala Ala Glu 290 295 300 gtt gta cat cct gga gac ctgaag aat tct gtt gaa gtc gca ctg aac 4381 Val Val His Pro Gly Asp Leu LysAsn Ser Val Glu Val Ala Leu Asn 305 310 315 aag ttg ctg gat cca atc cgggaa aag ttt aat acc cct gcc ctg aaa 4429 Lys Leu Leu Asp Pro Ile Arg GluLys Phe Asn Thr Pro Ala Leu Lys 320 325 330 aaa ctg gcc agc gct gcc taccca gat ccc tca aag cag aag cca atg 4477 Lys Leu Ala Ser Ala Ala Tyr ProAsp Pro Ser Lys Gln Lys Pro Met 335 340 345 350 gcc aaa ggc cct gcc aagaat tca gaa cca gag gag gtc atc cca tcc 4525 Ala Lys Gly Pro Ala Lys AsnSer Glu Pro Glu Glu Val Ile Pro Ser 355 360 365 cgg ctg gat atc cgt gtgggg aaa atc atc act gtg gag aag cac cca 4573 Arg Leu Asp Ile Arg Val GlyLys Ile Ile Thr Val Glu Lys His Pro 370 375 380 gat gca gac agc ctg tatgta gag aag att gac gtg ggg gaa gct gaa 4621 Asp Ala Asp Ser Leu Tyr ValGlu Lys Ile Asp Val Gly Glu Ala Glu 385 390 395 cca cgg act gtg gtg agcggc ctg gta cag ttc gtg ccc aag gag gaa 4669 Pro Arg Thr Val Val Ser GlyLeu Val Gln Phe Val Pro Lys Glu Glu 400 405 410 ctg cag gac agg ctg gtagtg gtg ctg tgc aac ctg aaa ccc cag aag 4717 Leu Gln Asp Arg Leu Val ValVal Leu Cys Asn Leu Lys Pro Gln Lys 415 420 425 430 atg aga gga gtc gagtcc caa ggc atg ctt ctg tgt gct tct ata gaa 4765 Met Arg Gly Val Glu SerGln Gly Met Leu Leu Cys Ala Ser Ile Glu 435 440 445 ggg ata aac cgc caggtt gaa cct ctg gac cct ccg gca ggc tct gct 4813 Gly Ile Asn Arg Gln ValGlu Pro Leu Asp Pro Pro Ala Gly Ser Ala 450 455 460 cct ggt gag cac gtgttt gtg aag ggc tat gaa aag ggc caa cca gat 4861 Pro Gly Glu His Val PheVal Lys Gly Tyr Glu Lys Gly Gln Pro Asp 465 470 475 gag gag ctc aag cccaag aag aaa gtc ttc gag aag ttg cag gct gac 4909 Glu Glu Leu Lys Pro LysLys Lys Val Phe Glu Lys Leu Gln Ala Asp 480 485 490 ttc aaa att tct gaggag tgc atc gca cag tgg aag caa acc aac ttc 4957 Phe Lys Ile Ser Glu GluCys Ile Ala Gln Trp Lys Gln Thr Asn Phe 495 500 505 510 atg acc aag ctgggc tcc att tcc tgt aaa tcg ctg aaa ggg ggg aac 5005 Met Thr Lys Leu GlySer Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn 515 520 525 att agc ctc gagcac cac cac cac cac cac tgagatccgg ctgctaacaa 5055 Ile Ser Leu Glu HisHis His His His His 530 535 agcccgaaag gaagctgagt tggctgctgc caccgctgagcaataactag cataacccct 5115 tggggcctct aaacgggtct tgaggggttt tttgctgaaaggaggaacta tatccggat 5174 2 536 PRT Artificial Sequence Description ofArtificial Sequence human full-length TyrRS in pET20B 2 Met Gly Asp AlaPro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15 Asn Leu GlnGlu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg GluLeu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His Val AlaTyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60 Ala Gly CysGlu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp AsnMet Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr GluAsn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu 100 105 110 GluLys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu 115 120 125Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130 135140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu 145150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu GluTyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg LysIle Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr SerLys Arg Val 195 200 205 His Leu Met Asn Pro Met Val Pro Gly Leu Thr GlySer Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu LeuAsp Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala PheCys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe IleLys His Val Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile Leu ArgAsp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala Tyr Val AspLeu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His Pro Gly Asp LeuLys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp ProIle Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala SerAla Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 GlyPro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375380 Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385390 395 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu LeuGln 405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln LysMet Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser IleGlu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala GlySer Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys Gly Tyr Glu Lys GlyGln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro Lys Lys Lys Val Phe GluLys Leu Gln Ala Asp Phe Lys 485 490 495 Ile Ser Glu Glu Cys Ile Ala GlnTrp Lys Gln Thr Asn Phe Met Thr 500 505 510 Lys Leu Gly Ser Ile Ser CysLys Ser Leu Lys Gly Gly Asn Ile Ser 515 520 525 Leu Glu His His His HisHis His 530 535 3 4682 DNA Artificial Sequence CDS (3428)..(4543)Description of Artificial Sequence human mini TyrRS in pET20B 3tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420tatacat atg ggg gac gct ccc agc cct gaa gag aaa ctg cac ctt atc 3469 MetGly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile 1 5 10 acc cgg aacctg cag gag gtt ctg ggg gaa gag aag ctg aag gag ata 3517 Thr Arg Asn LeuGln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile 15 20 25 30 ctg aag gagcgg gaa ctt aaa att tac tgg gga acg gca acc acg ggc 3565 Leu Lys Glu ArgGlu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly 35 40 45 aaa cca cat gtggct tac ttt gtg ccc atg tca aag att gca gac ttc 3613 Lys Pro His Val AlaTyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe 50 55 60 tta aag gca ggg tgtgag gta aca att ctg ttt gcg gac ctc cac gca 3661 Leu Lys Ala Gly Cys GluVal Thr Ile Leu Phe Ala Asp Leu His Ala 65 70 75 tac ctg gat aac atg aaagcc cca tgg gaa ctt cta gaa ctc cga gtc 3709 Tyr Leu Asp Asn Met Lys AlaPro Trp Glu Leu Leu Glu Leu Arg Val 80 85 90 agt tac tat gag aat gtg atcaaa gca atg ctg gag agc att ggt gtg 3757 Ser Tyr Tyr Glu Asn Val Ile LysAla Met Leu Glu Ser Ile Gly Val 95 100 105 110 ccc ttg gag aag ctc aagttc atc aaa ggc act gat tac cag ctc agc 3805 Pro Leu Glu Lys Leu Lys PheIle Lys Gly Thr Asp Tyr Gln Leu Ser 115 120 125 aaa gag tac aca cta gatgtg tac aga ctc tcc tcc gtg gtc aca cag 3853 Lys Glu Tyr Thr Leu Asp ValTyr Arg Leu Ser Ser Val Val Thr Gln 130 135 140 cac gat tcc aag aag gctgga gct gag gtg gta aag cag gtg gag cac 3901 His Asp Ser Lys Lys Ala GlyAla Glu Val Val Lys Gln Val Glu His 145 150 155 cct ttg ctg agt ggc ctctta tac ccc gga ctg cag gct ttg gat gaa 3949 Pro Leu Leu Ser Gly Leu LeuTyr Pro Gly Leu Gln Ala Leu Asp Glu 160 165 170 gag tat tta aaa gta gatgcc caa ttt gga ggc att gat cag aga aag 3997 Glu Tyr Leu Lys Val Asp AlaGln Phe Gly Gly Ile Asp Gln Arg Lys 175 180 185 190 att ttc acc ttt gcagag aag tac ctc cct gca ctt ggc tat tca aaa 4045 Ile Phe Thr Phe Ala GluLys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys 195 200 205 cgg gtc cat ctg atgaat cct atg gtt cca gga tta aca ggc agc aaa 4093 Arg Val His Leu Met AsnPro Met Val Pro Gly Leu Thr Gly Ser Lys 210 215 220 atg agc tct tca gaagag gag tcc aag att gat ctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu GluGlu Ser Lys Ile Asp Leu Leu Asp Arg Lys 225 230 235 gag gat gtg aag aaaaaa ctg aag aag gcc ttc tgt gag cca gga aat 4189 Glu Asp Val Lys Lys LysLeu Lys Lys Ala Phe Cys Glu Pro Gly Asn 240 245 250 gtg gag aac aat ggggtt ctg tcc ttc atc aag cat gtc ctt ttt ccc 4237 Val Glu Asn Asn Gly ValLeu Ser Phe Ile Lys His Val Leu Phe Pro 255 260 265 270 ctt aag tcc gagttt gtg atc cta cga gat gag aaa tgg ggt gga aac 4285 Leu Lys Ser Glu PheVal Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn 275 280 285 aaa acc tac acagct tac gtg gac ctg gaa aag gac ttt gct gct gag 4333 Lys Thr Tyr Thr AlaTyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu 290 295 300 gtt gta cat cctgga gac ctg aag aat tct gtt gaa gtc gca ctg aac 4381 Val Val His Pro GlyAsp Leu Lys Asn Ser Val Glu Val Ala Leu Asn 305 310 315 aag ttg ctg gatcca atc cgg gaa aag ttt aat acc cct gcc ctg aaa 4429 Lys Leu Leu Asp ProIle Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys 320 325 330 aaa ctg gcc agcgct gcc tac cca gat ccc tca aag cag aag cca atg 4477 Lys Leu Ala Ser AlaAla Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met 335 340 345 350 gcc aaa ggccct gcc aag aat tca gaa cca gag gag gtc atc ctc gag 4525 Ala Lys Gly ProAla Lys Asn Ser Glu Pro Glu Glu Val Ile Leu Glu 355 360 365 cac cac caccac cac cac tgagatccgg ctgctaacaa agcccgaaag 4573 His His His His HisHis 370 gaagctgagt tggctgctgc caccgctgag caataactag cataaccccttggggcctct 4633 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat4682 4 372 PRT Artificial Sequence Description of Artificial Sequencehuman mini TyrRS in pET20B 4 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys LeuHis Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu LysLeu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly ThrAla Thr Thr Gly Lys Pro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser LysIle Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe AlaAsp Leu His Ala Tyr Leu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu LeuLeu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala Met LeuGlu Ser Ile Gly Val Pro Leu 100 105 110 Glu Lys Leu Lys Phe Ile Lys GlyThr Asp Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr ArgLeu Ser Ser Val Val Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly AlaGlu Val Val Lys Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly LeuLeu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys ValAsp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr PheAla Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 HisLeu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu245 250 255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro LeuLys 260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly AsnLys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala AlaGlu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val AlaLeu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn ThrPro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro SerLys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys Asn Ser Glu ProGlu Glu Val Ile Leu Glu His His 355 360 365 His His His His 370 5 4100DNA Artificial Sequence CDS (3428)..(3961) Description of ArtificialSequence human TyrRS carboxyl-terminal domain in pET20B 5 tggcgaatgggacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgaccgctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgccacgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgatttagtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtgggccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagtggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgatttataagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaatttaacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaatgtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatgagacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaacatttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcacccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttacatcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgttttccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgccgggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactcaccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgccataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaaggagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaaccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatggcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaattaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccggctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcattgcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagtcaggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaagcattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcatttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatcccttaacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttcttgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctaccagcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttcagcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttcaagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgctgccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataaggcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacctacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaagggagaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggagcttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgacttgagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaacgcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcgttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgccgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatgcggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctcagtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtgactgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttgtctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtcagaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtggtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctccagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctgtttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgatgaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactggaacgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactcagggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagcatcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagactttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgcagcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggcaaccccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggccaggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggagaccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atgcca gag gag gtc atc cca tcc cgg ctg gat atc cgt gtg 3469 Met Pro Glu GluVal Ile Pro Ser Arg Leu Asp Ile Arg Val 1 5 10 ggg aaa atc atc act gtggag aag cac cca gat gca gac agc ctg tat 3517 Gly Lys Ile Ile Thr Val GluLys His Pro Asp Ala Asp Ser Leu Tyr 15 20 25 30 gta gag aag att gac gtgggg gaa gct gaa cca cgg act gtg gtg agc 3565 Val Glu Lys Ile Asp Val GlyGlu Ala Glu Pro Arg Thr Val Val Ser 35 40 45 ggc ctg gta cag ttc gtg cccaag gag gaa ctg cag gac agg ctg gta 3613 Gly Leu Val Gln Phe Val Pro LysGlu Glu Leu Gln Asp Arg Leu Val 50 55 60 gtg gtg ctg tgc aac ctg aaa ccccag aag atg aga gga gtc gag tcc 3661 Val Val Leu Cys Asn Leu Lys Pro GlnLys Met Arg Gly Val Glu Ser 65 70 75 caa ggc atg ctt ctg tgt gct tct atagaa ggg ata aac cgc cag gtt 3709 Gln Gly Met Leu Leu Cys Ala Ser Ile GluGly Ile Asn Arg Gln Val 80 85 90 gaa cct ctg gac cct ccg gca ggc tct gctcct ggt gag cac gtg ttt 3757 Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala ProGly Glu His Val Phe 95 100 105 110 gtg aag ggc tat gaa aag ggc caa ccagat gag gag ctc aag ccc aag 3805 Val Lys Gly Tyr Glu Lys Gly Gln Pro AspGlu Glu Leu Lys Pro Lys 115 120 125 aag aaa gtc ttc gag aag ttg cag gctgac ttc aaa att tct gag gag 3853 Lys Lys Val Phe Glu Lys Leu Gln Ala AspPhe Lys Ile Ser Glu Glu 130 135 140 tgc atc gca cag tgg aag caa acc aacttc atg acc aag ctg ggc tcc 3901 Cys Ile Ala Gln Trp Lys Gln Thr Asn PheMet Thr Lys Leu Gly Ser 145 150 155 att tcc tgt aaa tcg ctg aaa ggg gggaac att agc ctc gag cac cac 3949 Ile Ser Cys Lys Ser Leu Lys Gly Gly AsnIle Ser Leu Glu His His 160 165 170 cac cac cac cac tgagatccggctgctaacaa agcccgaaag gaagctgagt 4001 His His His His 175 tggctgctgccaccgctgag caataactag cataacccct tggggcctct aaacgggtct 4061 tgaggggttttttgctgaaa ggaggaacta tatccggat 4100 6 178 PRT Artificial SequenceDescription of Artificial Sequence human TyrRS carboxyl-terminal domainin pET20B 6 Met Pro Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val GlyLys 1 5 10 15 Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu TyrVal Glu 20 25 30 Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val Val SerGly Leu 35 40 45 Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu ValVal Val 50 55 60 Leu Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val Glu SerGln Gly 65 70 75 80 Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg GlnVal Glu Pro 85 90 95 Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu His ValPhe Val Lys 100 105 110 Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu LysPro Lys Lys Lys 115 120 125 Val Phe Glu Lys Leu Gln Ala Asp Phe Lys IleSer Glu Glu Cys Ile 130 135 140 Ala Gln Trp Lys Gln Thr Asn Phe Met ThrLys Leu Gly Ser Ile Ser 145 150 155 160 Cys Lys Ser Leu Lys Gly Gly AsnIle Ser Leu Glu His His His His 165 170 175 His His 7 4682 DNAArtificial Sequence CDS (3428)..(4543) Description of ArtificialSequence human mini TyrRS mutant in pET20B 7 tggcgaatgg gacgcgccctgtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttgccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccggctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttacggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccctgatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgttccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattttgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaattttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaacccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataaccctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtgtcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgctggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactggatctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatgagcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagcaactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacagaaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatgagtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccgcttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctgaatgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgttgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagactggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggtttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactggggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaactatggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaactgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaatttaaaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagttttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggtttgtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgcagataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctgtagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcgataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggtcgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaactgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcggacaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggggaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgatttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttttacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctgattctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaacgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttctccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctgctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatggctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccggcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttcaccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagcgattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgttaatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcactgatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagagaggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgagggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgccagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatgcagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaacacggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcgcttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccagcctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacgctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggtttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg ggg gac gctccc agc cct gaa gag aaa ctg cac ctt atc 3469 Met Gly Asp Ala Pro Ser ProGlu Glu Lys Leu His Leu Ile 1 5 10 acc cgg aac ctg cag gag gtt ctg ggggaa gag aag ctg aag gag ata 3517 Thr Arg Asn Leu Gln Glu Val Leu Gly GluGlu Lys Leu Lys Glu Ile 15 20 25 30 ctg aag gag cgg gaa ctt aaa att tactgg gga acg gca acc acg ggc 3565 Leu Lys Glu Arg Glu Leu Lys Ile Tyr TrpGly Thr Ala Thr Thr Gly 35 40 45 aaa cca cat gtg gct tac ttt gtg ccc atgtca aag att gca gac ttc 3613 Lys Pro His Val Ala Tyr Phe Val Pro Met SerLys Ile Ala Asp Phe 50 55 60 tta aag gca ggg tgt gag gta aca att ctg tttgcg gac ctc cac gca 3661 Leu Lys Ala Gly Cys Glu Val Thr Ile Leu Phe AlaAsp Leu His Ala 65 70 75 tac ctg gat aac atg aaa gcc cca tgg gaa ctt ctagaa ctg cag gtc 3709 Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu GluLeu Gln Val 80 85 90 agt tac tat gag aat gtg atc aaa gca atg ctg gag agcatt ggt gtg 3757 Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser IleGly Val 95 100 105 110 ccc ttg gag aag ctc aag ttc atc aaa ggc act gattac cag ctc agc 3805 Pro Leu Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp TyrGln Leu Ser 115 120 125 aaa gag tac aca cta gat gtg tac aga ctc tcc tccgtg gtc aca cag 3853 Lys Glu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser ValVal Thr Gln 130 135 140 cac gat tcc aag aag gct gga gct gag gtg gta aagcag gtg gag cac 3901 His Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys GlnVal Glu His 145 150 155 cct ttg ctg agt ggc ctc tta tac ccc gga ctg caggct ttg gat gaa 3949 Pro Leu Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln AlaLeu Asp Glu 160 165 170 gag tat tta aaa gta gat gcc caa ttt gga ggc attgat cag aga aag 3997 Glu Tyr Leu Lys Val Asp Ala Gln Phe Gly Gly Ile AspGln Arg Lys 175 180 185 190 att ttc acc ttt gca gag aag tac ctc cct gcactt ggc tat tca aaa 4045 Ile Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala LeuGly Tyr Ser Lys 195 200 205 cgg gtc cat ctg atg aat cct atg gtt cca ggatta aca ggc agc aaa 4093 Arg Val His Leu Met Asn Pro Met Val Pro Gly LeuThr Gly Ser Lys 210 215 220 atg agc tct tca gaa gag gag tcc aag att gatctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp LeuLeu Asp Arg Lys 225 230 235 gag gat gtg aag aaa aaa ctg aag aag gcc ttctgt gag cca gga aat 4189 Glu Asp Val Lys Lys Lys Leu Lys Lys Ala Phe CysGlu Pro Gly Asn 240 245 250 gtg gag aac aat ggg gtt ctg tcc ttc atc aagcat gtc ctt ttt ccc 4237 Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys HisVal Leu Phe Pro 255 260 265 270 ctt aag tcc gag ttt gtg atc cta cga gatgag aaa tgg ggt gga aac 4285 Leu Lys Ser Glu Phe Val Ile Leu Arg Asp GluLys Trp Gly Gly Asn 275 280 285 aaa acc tac aca gct tac gtg gac ctg gaaaag gac ttt gct gct gag 4333 Lys Thr Tyr Thr Ala Tyr Val Asp Leu Glu LysAsp Phe Ala Ala Glu 290 295 300 gtt gta cat cct gga gac ctg aag aat tctgtt gaa gtc gca ctg aac 4381 Val Val His Pro Gly Asp Leu Lys Asn Ser ValGlu Val Ala Leu Asn 305 310 315 aag ttg ctg gat cca atc cgg gaa aag tttaat acc cct gcc ctg aaa 4429 Lys Leu Leu Asp Pro Ile Arg Glu Lys Phe AsnThr Pro Ala Leu Lys 320 325 330 aaa ctg gcc agc gct gcc tac cca gat ccctca aag cag aag cca atg 4477 Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro SerLys Gln Lys Pro Met 335 340 345 350 gcc aaa ggc cct gcc aag aat tca gaacca gag gag gtc atc ctc gag 4525 Ala Lys Gly Pro Ala Lys Asn Ser Glu ProGlu Glu Val Ile Leu Glu 355 360 365 cac cac cac cac cac cac tgagatccggctgctaacaa agcccgaaag 4573 His His His His His His 370 gaagctgagttggctgctgc caccgctgag caataactag cataacccct tggggcctct 4633 aaacgggtcttgaggggttt tttgctgaaa ggaggaacta tatccggat 4682 8 372 PRT ArtificialSequence Description of Artificial Sequence human mini TyrRS mutant inpET20B 8 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile LeuLys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly LysPro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe LeuLys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala TyrLeu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Gln ValSer Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly ValPro Leu 100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln LeuSer Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val ValThr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys GlnVal Glu His Pro Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly LeuGln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe GlyGly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr LeuPro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro MetVal Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu GluSer Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys LysLys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn AsnGly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270 SerGlu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys LysLeu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro MetAla Lys 340 345 350 Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile LeuGlu His His 355 360 365 His His His His 370 9 5018 DNA ArtificialSequence CDS (3428)..(4879) Description of Artificial Sequence humanfull-length TrpRS in pET20B 9 tggcgaatgg gacgcgccct gtagcggcgcattaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccctagcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccgtcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcgaccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggtttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactggaacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttcggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaatattaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttgtttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaatgcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttattccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagtaaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacagcggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaaagttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcgccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatcttacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacactgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgcacaacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccataccaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaactattaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggcggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctgataaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatggtaagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacgaaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagaccaagtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatctaggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttccactgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcgcgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccggatcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaatactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcctacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtgtcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaacggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacctacagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatccggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctggtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatgctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcctggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtggataaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcgcagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgcatctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgccgcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgccccgacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgcttacagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcaccgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacagatgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggcttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccgtgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctcacgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaactggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcgttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccggaacataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccgaagaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttcgctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccgggtcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgagatctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctagaaataatttt gtttaacttt aagaaggaga 3420 tatacat atg ccc aac agt gag cccgca tct ctg ctg gag ctg ttc aac 3469 Met Pro Asn Ser Glu Pro Ala Ser LeuLeu Glu Leu Phe Asn 1 5 10 agc atc gcc aca caa ggg gag ctc gta agg tccctc aaa gcg gga aat 3517 Ser Ile Ala Thr Gln Gly Glu Leu Val Arg Ser LeuLys Ala Gly Asn 15 20 25 30 gcg tca aag gat gaa att gat tct gca gta aagatg ttg gtg tca tta 3565 Ala Ser Lys Asp Glu Ile Asp Ser Ala Val Lys MetLeu Val Ser Leu 35 40 45 aaa atg agc tac aaa gct gcc gcg ggg gag gat tacaag gct gac tgt 3613 Lys Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr LysAla Asp Cys 50 55 60 cct cca ggg aac cca gca cct acc agt aat cat ggc ccagat gcc aca 3661 Pro Pro Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro AspAla Thr 65 70 75 gaa gct gaa gag gat ttt gtg gac cca tgg aca gta cag acaagc agt 3709 Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr SerSer 80 85 90 gca aaa ggc ata gac tac gat aag ctc att gtt cgg ttt gga agtagt 3757 Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser95 100 105 110 aaa att gac aaa gag cta ata aac cga ata gag aga gcc accggc caa 3805 Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr GlyGln 115 120 125 aga cca cac cac ttc ctg cgc aga ggc atc ttc ttc tca cacaga gat 3853 Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His ArgAsp 130 135 140 atg aat cag gtt ctt gat gcc tat gaa aat aag aag cca ttttat ctg 3901 Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe TyrLeu 145 150 155 tac acg ggc cgg ggc ccc tct tct gaa gca atg cat gta ggtcac ctc 3949 Tyr Thr Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly HisLeu 160 165 170 att cca ttt att ttc aca aag tgg ctc cag gat gta ttt aacgtg ccc 3997 Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn ValPro 175 180 185 190 ttg gtc atc cag atg acg gat gac gag aag tat ctg tggaag gac ctg 4045 Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp LysAsp Leu 195 200 205 acc ctg gac cag gcc tat ggc gat gct gtt gag aat gccaag gac atc 4093 Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala LysAsp Ile 210 215 220 atc gcc tgt ggc ttt gac atc aac aag act ttc ata ttctct gac ctg 4141 Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe SerAsp Leu 225 230 235 gac tac atg ggg atg agc tca ggt ttc tac aaa aat gtggtg aag att 4189 Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val ValLys Ile 240 245 250 caa aag cat gtt acc ttc aac caa gtg aaa ggc att ttcggc ttc act 4237 Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe GlyPhe Thr 255 260 265 270 gac agc gac tgc att ggg aag atc agt ttt cct gccatc cag gct gct 4285 Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala IleGln Ala Ala 275 280 285 ccc tcc ttc agc aac tca ttc cca cag atc ttc cgagac agg acg gat 4333 Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg AspArg Thr Asp 290 295 300 atc cag tgc ctt atc cca tgt gcc att gac cag gatcct tac ttt aga 4381 Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp ProTyr Phe Arg 305 310 315 atg aca agg gac gtc gcc ccc agg atc ggc tat cctaaa cca gcc ctg 4429 Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro LysPro Ala Leu 320 325 330 ttg cac tcc acc ttc ttc cca gcc ctg cag ggc gcccag acc aaa atg 4477 Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala GlnThr Lys Met 335 340 345 350 agt gcc agc gac cca aac tcc tcc atc ttc ctcacc gac acg gcc aag 4525 Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu ThrAsp Thr Ala Lys 355 360 365 cag atc aaa acc aag gtc aat aag cat gcg ttttct gga ggg aga gac 4573 Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe SerGly Gly Arg Asp 370 375 380 acc atc gag gag cac agg cag ttt ggg ggc aactgt gat gtg gac gtg 4621 Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn CysAsp Val Asp Val 385 390 395 tct ttc atg tac ctg acc ttc ttc ctc gag gacgac gac aag ctc gag 4669 Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp AspAsp Lys Leu Glu 400 405 410 cag atc agg aag gat tac acc agc gga gcc atgctc acc ggt gag ctc 4717 Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met LeuThr Gly Glu Leu 415 420 425 430 aag aag gca ctc ata gag gtt ctg cag cccttg atc gca gag cac cag 4765 Lys Lys Ala Leu Ile Glu Val Leu Gln Pro LeuIle Ala Glu His Gln 435 440 445 gcc cgg cgc aag gag gtc acg gat gag atagtg aaa gag ttc atg act 4813 Ala Arg Arg Lys Glu Val Thr Asp Glu Ile ValLys Glu Phe Met Thr 450 455 460 ccc cgg aag ctg tcc ttc gac ttt cag aagctt gcg gcc gca ctc gag 4861 Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys LeuAla Ala Ala Leu Glu 465 470 475 cac cac cac cac cac cac tgagatccggctgctaacaa agcccgaaag 4909 His His His His His His 480 gaagctgagttggctgctgc caccgctgag caataactag cataacccct tggggcctct 4969 aaacgggtcttgaggggttt tttgctgaaa ggaggaacta tatccggat 5018 10 484 PRT ArtificialSequence Description of Artificial Sequence human full-length TrpRS inpET20B 10 Met Pro Asn Ser Glu Pro Ala Ser Leu Leu Glu Leu Phe Asn SerIle 1 5 10 15 Ala Thr Gln Gly Glu Leu Val Arg Ser Leu Lys Ala Gly AsnAla Ser 20 25 30 Lys Asp Glu Ile Asp Ser Ala Val Lys Met Leu Val Ser LeuLys Met 35 40 45 Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp CysPro Pro 50 55 60 Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala ThrGlu Ala 65 70 75 80 Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr SerSer Ala Lys 85 90 95 Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly SerSer Lys Ile 100 105 110 Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala ThrGly Gln Arg Pro 115 120 125 His His Phe Leu Arg Arg Gly Ile Phe Phe SerHis Arg Asp Met Asn 130 135 140 Gln Val Leu Asp Ala Tyr Glu Asn Lys LysPro Phe Tyr Leu Tyr Thr 145 150 155 160 Gly Arg Gly Pro Ser Ser Glu AlaMet His Val Gly His Leu Ile Pro 165 170 175 Phe Ile Phe Thr Lys Trp LeuGln Asp Val Phe Asn Val Pro Leu Val 180 185 190 Ile Gln Met Thr Asp AspGlu Lys Tyr Leu Trp Lys Asp Leu Thr Leu 195 200 205 Asp Gln Ala Tyr GlyAsp Ala Val Glu Asn Ala Lys Asp Ile Ile Ala 210 215 220 Cys Gly Phe AspIle Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr 225 230 235 240 Met GlyMet Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys 245 250 255 HisVal Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr Asp Ser 260 265 270Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser 275 280285 Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln 290295 300 Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr305 310 315 320 Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala LeuLeu His 325 330 335 Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr LysMet Ser Ala 340 345 350 Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp ThrAla Lys Gln Ile 355 360 365 Lys Thr Lys Val Asn Lys His Ala Phe Ser GlyGly Arg Asp Thr Ile 370 375 380 Glu Glu His Arg Gln Phe Gly Gly Asn CysAsp Val Asp Val Ser Phe 385 390 395 400 Met Tyr Leu Thr Phe Phe Leu GluAsp Asp Asp Lys Leu Glu Gln Ile 405 410 415 Arg Lys Asp Tyr Thr Ser GlyAla Met Leu Thr Gly Glu Leu Lys Lys 420 425 430 Ala Leu Ile Glu Val LeuGln Pro Leu Ile Ala Glu His Gln Ala Arg 435 440 445 Arg Lys Glu Val ThrAsp Glu Ile Val Lys Glu Phe Met Thr Pro Arg 450 455 460 Lys Leu Ser PheAsp Phe Gln Lys Leu Ala Ala Ala Leu Glu His His 465 470 475 480 His HisHis His 11 4877 DNA Artificial Sequence CDS (3428)..(4738) Descriptionof Artificial Sequence human mini TrpRS in pET20B 11 tggcgaatgggacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgaccgctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgccacgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgatttagtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtgggccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagtggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgatttataagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaatttaacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaatgtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatgagacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaacatttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcacccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttacatcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgttttccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgccgggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactcaccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgccataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaaggagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaaccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatggcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaattaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccggctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcattgcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagtcaggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaagcattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcatttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatcccttaacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttcttgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctaccagcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttcagcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttcaagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgctgccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataaggcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacctacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaagggagaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggagcttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgacttgagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaacgcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcgttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgccgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatgcggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctcagtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtgactgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttgtctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtcagaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtggtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctccagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctgtttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgatgaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactggaacgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactcagggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagcatcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagactttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgcagcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggcaaccccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggccaggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggagaccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atgagc tac aaa gct gcc gcg ggg gag gat tac aag gct gac 3469 Met Ser Tyr LysAla Ala Ala Gly Glu Asp Tyr Lys Ala Asp 1 5 10 tgt cct cca ggg aac ccagca cct acc agt aat cat ggc cca gat gcc 3517 Cys Pro Pro Gly Asn Pro AlaPro Thr Ser Asn His Gly Pro Asp Ala 15 20 25 30 aca gaa gct gaa gag gatttt gtg gac cca tgg aca gta cag aca agc 3565 Thr Glu Ala Glu Glu Asp PheVal Asp Pro Trp Thr Val Gln Thr Ser 35 40 45 agt gca aaa ggc ata gac tacgat aag ctc att gtt cgg ttt gga agt 3613 Ser Ala Lys Gly Ile Asp Tyr AspLys Leu Ile Val Arg Phe Gly Ser 50 55 60 agt aaa att gac aaa gag cta ataaac cga ata gag aga gcc acc ggc 3661 Ser Lys Ile Asp Lys Glu Leu Ile AsnArg Ile Glu Arg Ala Thr Gly 65 70 75 caa aga cca cac cac ttc ctg cgc agaggc atc ttc ttc tca cac aga 3709 Gln Arg Pro His His Phe Leu Arg Arg GlyIle Phe Phe Ser His Arg 80 85 90 gat atg aat cag gtt ctt gat gcc tat gaaaat aag aag cca ttt tat 3757 Asp Met Asn Gln Val Leu Asp Ala Tyr Glu AsnLys Lys Pro Phe Tyr 95 100 105 110 ctg tac acg ggc cgg ggc ccc tct tctgaa gca atg cat gta ggt cac 3805 Leu Tyr Thr Gly Arg Gly Pro Ser Ser GluAla Met His Val Gly His 115 120 125 ctc att cca ttt att ttc aca aag tggctc cag gat gta ttt aac gtg 3853 Leu Ile Pro Phe Ile Phe Thr Lys Trp LeuGln Asp Val Phe Asn Val 130 135 140 ccc ttg gtc atc cag atg acg gat gacgag aag tat ctg tgg aag gac 3901 Pro Leu Val Ile Gln Met Thr Asp Asp GluLys Tyr Leu Trp Lys Asp 145 150 155 ctg acc ctg gac cag gcc tat ggc gatgct gtt gag aat gcc aag gac 3949 Leu Thr Leu Asp Gln Ala Tyr Gly Asp AlaVal Glu Asn Ala Lys Asp 160 165 170 atc atc gcc tgt ggc ttt gac atc aacaag act ttc ata ttc tct gac 3997 Ile Ile Ala Cys Gly Phe Asp Ile Asn LysThr Phe Ile Phe Ser Asp 175 180 185 190 ctg gac tac atg ggg atg agc tcaggt ttc tac aaa aat gtg gtg aag 4045 Leu Asp Tyr Met Gly Met Ser Ser GlyPhe Tyr Lys Asn Val Val Lys 195 200 205 att caa aag cat gtt acc ttc aaccaa gtg aaa ggc att ttc ggc ttc 4093 Ile Gln Lys His Val Thr Phe Asn GlnVal Lys Gly Ile Phe Gly Phe 210 215 220 act gac agc gac tgc att ggg aagatc agt ttt cct gcc atc cag gct 4141 Thr Asp Ser Asp Cys Ile Gly Lys IleSer Phe Pro Ala Ile Gln Ala 225 230 235 gct ccc tcc ttc agc aac tca ttccca cag atc ttc cga gac agg acg 4189 Ala Pro Ser Phe Ser Asn Ser Phe ProGln Ile Phe Arg Asp Arg Thr 240 245 250 gat atc cag tgc ctt atc cca tgtgcc att gac cag gat cct tac ttt 4237 Asp Ile Gln Cys Leu Ile Pro Cys AlaIle Asp Gln Asp Pro Tyr Phe 255 260 265 270 aga atg aca agg gac gtc gccccc agg atc ggc tat cct aaa cca gcc 4285 Arg Met Thr Arg Asp Val Ala ProArg Ile Gly Tyr Pro Lys Pro Ala 275 280 285 ctg ttg cac tcc acc ttc ttccca gcc ctg cag ggc gcc cag acc aaa 4333 Leu Leu His Ser Thr Phe Phe ProAla Leu Gln Gly Ala Gln Thr Lys 290 295 300 atg agt gcc agc gac cca aactcc tcc atc ttc ctc acc gac acg gcc 4381 Met Ser Ala Ser Asp Pro Asn SerSer Ile Phe Leu Thr Asp Thr Ala 305 310 315 aag cag atc aaa acc aag gtcaat aag cat gcg ttt tct gga ggg aga 4429 Lys Gln Ile Lys Thr Lys Val AsnLys His Ala Phe Ser Gly Gly Arg 320 325 330 gac acc atc gag gag cac aggcag ttt ggg ggc aac tgt gat gtg gac 4477 Asp Thr Ile Glu Glu His Arg GlnPhe Gly Gly Asn Cys Asp Val Asp 335 340 345 350 gtg tct ttc atg tac ctgacc ttc ttc ctc gag gac gac gac aag ctc 4525 Val Ser Phe Met Tyr Leu ThrPhe Phe Leu Glu Asp Asp Asp Lys Leu 355 360 365 gag cag atc agg aag gattac acc agc gga gcc atg ctc acc ggt gag 4573 Glu Gln Ile Arg Lys Asp TyrThr Ser Gly Ala Met Leu Thr Gly Glu 370 375 380 ctc aag aag gca ctc atagag gtt ctg cag ccc ttg atc gca gag cac 4621 Leu Lys Lys Ala Leu Ile GluVal Leu Gln Pro Leu Ile Ala Glu His 385 390 395 cag gcc cgg cgc aag gaggtc acg gat gag ata gtg aaa gag ttc atg 4669 Gln Ala Arg Arg Lys Glu ValThr Asp Glu Ile Val Lys Glu Phe Met 400 405 410 act ccc cgg aag ctg tccttc gac ttt cag aag ctt gcg gcc gca ctc 4717 Thr Pro Arg Lys Leu Ser PheAsp Phe Gln Lys Leu Ala Ala Ala Leu 415 420 425 430 gag cac cac cac caccac cac tgagatccgg ctgctaacaa agcccgaaag 4768 Glu His His His His HisHis 435 gaagctgagt tggctgctgc caccgctgag caataactag cataaccccttggggcctct 4828 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat4877 12 437 PRT Artificial Sequence Description of Artificial Sequencehuman mini TrpRS in pET20B 12 Met Ser Tyr Lys Ala Ala Ala Gly Glu AspTyr Lys Ala Asp Cys Pro 1 5 10 15 Pro Gly Asn Pro Ala Pro Thr Ser AsnHis Gly Pro Asp Ala Thr Glu 20 25 30 Ala Glu Glu Asp Phe Val Asp Pro TrpThr Val Gln Thr Ser Ser Ala 35 40 45 Lys Gly Ile Asp Tyr Asp Lys Leu IleVal Arg Phe Gly Ser Ser Lys 50 55 60 Ile Asp Lys Glu Leu Ile Asn Arg IleGlu Arg Ala Thr Gly Gln Arg 65 70 75 80 Pro His His Phe Leu Arg Arg GlyIle Phe Phe Ser His Arg Asp Met 85 90 95 Asn Gln Val Leu Asp Ala Tyr GluAsn Lys Lys Pro Phe Tyr Leu Tyr 100 105 110 Thr Gly Arg Gly Pro Ser SerGlu Ala Met His Val Gly His Leu Ile 115 120 125 Pro Phe Ile Phe Thr LysTrp Leu Gln Asp Val Phe Asn Val Pro Leu 130 135 140 Val Ile Gln Met ThrAsp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr 145 150 155 160 Leu Asp GlnAla Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp Ile Ile 165 170 175 Ala CysGly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp 180 185 190 TyrMet Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln 195 200 205Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr Asp 210 215220 Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro 225230 235 240 Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr AspIle 245 250 255 Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr PheArg Met 260 265 270 Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys ProAla Leu Leu 275 280 285 His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala GlnThr Lys Met Ser 290 295 300 Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu ThrAsp Thr Ala Lys Gln 305 310 315 320 Ile Lys Thr Lys Val Asn Lys His AlaPhe Ser Gly Gly Arg Asp Thr 325 330 335 Ile Glu Glu His Arg Gln Phe GlyGly Asn Cys Asp Val Asp Val Ser 340 345 350 Phe Met Tyr Leu Thr Phe PheLeu Glu Asp Asp Asp Lys Leu Glu Gln 355 360 365 Ile Arg Lys Asp Tyr ThrSer Gly Ala Met Leu Thr Gly Glu Leu Lys 370 375 380 Lys Ala Leu Ile GluVal Leu Gln Pro Leu Ile Ala Glu His Gln Ala 385 390 395 400 Arg Arg LysGlu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro 405 410 415 Arg LysLeu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu His 420 425 430 HisHis His His His 435 13 4811 DNA Artificial Sequence CDS (3428)..(4672)Description of Artificial Sequence human supermini TrpRS in pET20B 13tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420tatacat atg agt aat cat ggc cca gat gcc aca gaa gct gaa gag gat 3469 MetSer Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp 1 5 10 ttt gtg gaccca tgg aca gta cag aca agc agt gca aaa ggc ata gac 3517 Phe Val Asp ProTrp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp 15 20 25 30 tac gat aagctc att gtt cgg ttt gga agt agt aaa att gac aaa gag 3565 Tyr Asp Lys LeuIle Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu 35 40 45 cta ata aac cgaata gag aga gcc acc ggc caa aga cca cac cac ttc 3613 Leu Ile Asn Arg IleGlu Arg Ala Thr Gly Gln Arg Pro His His Phe 50 55 60 ctg cgc aga ggc atcttc ttc tca cac aga gat atg aat cag gtt ctt 3661 Leu Arg Arg Gly Ile PhePhe Ser His Arg Asp Met Asn Gln Val Leu 65 70 75 gat gcc tat gaa aat aagaag cca ttt tat ctg tac acg ggc cgg ggc 3709 Asp Ala Tyr Glu Asn Lys LysPro Phe Tyr Leu Tyr Thr Gly Arg Gly 80 85 90 ccc tct tct gaa gca atg catgta ggt cac ctc att cca ttt att ttc 3757 Pro Ser Ser Glu Ala Met His ValGly His Leu Ile Pro Phe Ile Phe 95 100 105 110 aca aag tgg ctc cag gatgta ttt aac gtg ccc ttg gtc atc cag atg 3805 Thr Lys Trp Leu Gln Asp ValPhe Asn Val Pro Leu Val Ile Gln Met 115 120 125 acg gat gac gag aag tatctg tgg aag gac ctg acc ctg gac cag gcc 3853 Thr Asp Asp Glu Lys Tyr LeuTrp Lys Asp Leu Thr Leu Asp Gln Ala 130 135 140 tat ggc gat gct gtt gagaat gcc aag gac atc atc gcc tgt ggc ttt 3901 Tyr Gly Asp Ala Val Glu AsnAla Lys Asp Ile Ile Ala Cys Gly Phe 145 150 155 gac atc aac aag act ttcata ttc tct gac ctg gac tac atg ggg atg 3949 Asp Ile Asn Lys Thr Phe IlePhe Ser Asp Leu Asp Tyr Met Gly Met 160 165 170 agc tca ggt ttc tac aaaaat gtg gtg aag att caa aag cat gtt acc 3997 Ser Ser Gly Phe Tyr Lys AsnVal Val Lys Ile Gln Lys His Val Thr 175 180 185 190 ttc aac caa gtg aaaggc att ttc ggc ttc act gac agc gac tgc att 4045 Phe Asn Gln Val Lys GlyIle Phe Gly Phe Thr Asp Ser Asp Cys Ile 195 200 205 ggg aag atc agt tttcct gcc atc cag gct gct ccc tcc ttc agc aac 4093 Gly Lys Ile Ser Phe ProAla Ile Gln Ala Ala Pro Ser Phe Ser Asn 210 215 220 tca ttc cca cag atcttc cga gac agg acg gat atc cag tgc ctt atc 4141 Ser Phe Pro Gln Ile PheArg Asp Arg Thr Asp Ile Gln Cys Leu Ile 225 230 235 cca tgt gcc att gaccag gat cct tac ttt aga atg aca agg gac gtc 4189 Pro Cys Ala Ile Asp GlnAsp Pro Tyr Phe Arg Met Thr Arg Asp Val 240 245 250 gcc ccc agg atc ggctat cct aaa cca gcc ctg ttg cac tcc acc ttc 4237 Ala Pro Arg Ile Gly TyrPro Lys Pro Ala Leu Leu His Ser Thr Phe 255 260 265 270 ttc cca gcc ctgcag ggc gcc cag acc aaa atg agt gcc agc gac cca 4285 Phe Pro Ala Leu GlnGly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro 275 280 285 aac tcc tcc atcttc ctc acc gac acg gcc aag cag atc aaa acc aag 4333 Asn Ser Ser Ile PheLeu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys 290 295 300 gtc aat aag catgcg ttt tct gga ggg aga gac acc atc gag gag cac 4381 Val Asn Lys His AlaPhe Ser Gly Gly Arg Asp Thr Ile Glu Glu His 305 310 315 agg cag ttt gggggc aac tgt gat gtg gac gtg tct ttc atg tac ctg 4429 Arg Gln Phe Gly GlyAsn Cys Asp Val Asp Val Ser Phe Met Tyr Leu 320 325 330 acc ttc ttc ctcgag gac gac gac aag ctc gag cag atc agg aag gat 4477 Thr Phe Phe Leu GluAsp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp 335 340 345 350 tac acc agcgga gcc atg ctc acc ggt gag ctc aag aag gca ctc ata 4525 Tyr Thr Ser GlyAla Met Leu Thr Gly Glu Leu Lys Lys Ala Leu Ile 355 360 365 gag gtt ctgcag ccc ttg atc gca gag cac cag gcc cgg cgc aag gag 4573 Glu Val Leu GlnPro Leu Ile Ala Glu His Gln Ala Arg Arg Lys Glu 370 375 380 gtc acg gatgag ata gtg aaa gag ttc atg act ccc cgg aag ctg tcc 4621 Val Thr Asp GluIle Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser 385 390 395 ttc gac tttcag aag ctt gcg gcc gca ctc gag cac cac cac cac cac 4669 Phe Asp Phe GlnLys Leu Ala Ala Ala Leu Glu His His His His His 400 405 410 cactgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 4722 His 415caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 4782tttgctgaaa ggaggaacta tatccggat 4811 14 415 PRT Artificial SequenceDescription of Artificial Sequence human supermini TrpRS in pET20B 14Met Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val 1 5 1015 Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp 20 2530 Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile 35 4045 Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu Arg 50 5560 Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp Ala 65 7075 80 Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr Gly Arg Gly Pro Ser 8590 95 Ser Glu Ala Met His Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys100 105 110 Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met ThrAsp 115 120 125 Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu Asp Gln AlaTyr Gly 130 135 140 Asp Ala Val Glu Asn Ala Lys Asp Ile Ile Ala Cys GlyPhe Asp Ile 145 150 155 160 Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp TyrMet Gly Met Ser Ser 165 170 175 Gly Phe Tyr Lys Asn Val Val Lys Ile GlnLys His Val Thr Phe Asn 180 185 190 Gln Val Lys Gly Ile Phe Gly Phe ThrAsp Ser Asp Cys Ile Gly Lys 195 200 205 Ile Ser Phe Pro Ala Ile Gln AlaAla Pro Ser Phe Ser Asn Ser Phe 210 215 220 Pro Gln Ile Phe Arg Asp ArgThr Asp Ile Gln Cys Leu Ile Pro Cys 225 230 235 240 Ala Ile Asp Gln AspPro Tyr Phe Arg Met Thr Arg Asp Val Ala Pro 245 250 255 Arg Ile Gly TyrPro Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro 260 265 270 Ala Leu GlnGly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser 275 280 285 Ser IlePhe Leu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn 290 295 300 LysHis Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln 305 310 315320 Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr Leu Thr Phe 325330 335 Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr340 345 350 Ser Gly Ala Met Leu Thr Gly Glu Leu Lys Lys Ala Leu Ile GluVal 355 360 365 Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg Arg Lys GluVal Thr 370 375 380 Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg Lys LeuSer Phe Asp 385 390 395 400 Phe Gln Lys Leu Ala Ala Ala Leu Glu His HisHis His His His 405 410 415 15 4742 DNA Artificial Sequence CDS(3428)..(4603) Description of Artificial Sequence human minor Trp-RSfragment in pET20B 15 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcggcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgctcctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctctaaatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaacttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccctttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactcaaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattggttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgtttacaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttctaaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataatattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tcccttttttgcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgctgaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatccttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgctatgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacactattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggcatgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaacttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggggatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgacgagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggcgaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagttgcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctggagccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcccgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacagatcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactcatatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatcctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtcagaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgctgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagctaccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtccttctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctcgctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccgggttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcgtgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgagctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggcagggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttatagtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggggggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgctggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtattaccgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtcagtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggtatttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaagccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgccaacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagctgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgcgaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctgttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaagcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaagggggatttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggttactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatggatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacagatgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggtgcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattcatgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtatcggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacgacaggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatcccgcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataattttgtttaacttt aagaaggaga 3420 tatacat atg agt gca aaa ggc ata gac tac gataag ctc att gtt cgg 3469 Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu IleVal Arg 1 5 10 ttt gga agt agt aaa att gac aaa gag cta ata aac cga atagag aga 3517 Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile GluArg 15 20 25 30 gcc acc ggc caa aga cca cac cac ttc ctg cgc aga ggc atcttc ttc 3565 Ala Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile PhePhe 35 40 45 tca cac aga gat atg aat cag gtt ctt gat gcc tat gaa aat aagaag 3613 Ser His Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys50 55 60 cca ttt tat ctg tac acg ggc cgg ggc ccc tct tct gaa gca atg cat3661 Pro Phe Tyr Leu Tyr Thr Gly Arg Gly Pro Ser Ser Glu Ala Met His 6570 75 gta ggt cac ctc att cca ttt att ttc aca aag tgg ctc cag gat gta3709 Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val 8085 90 ttt aac gtg ccc ttg gtc atc cag atg acg gat gac gag aag tat ctg3757 Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu 95100 105 110 tgg aag gac ctg acc ctg gac cag gcc tat ggc gat gct gtt gagaat 3805 Trp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn115 120 125 gcc aag gac atc atc gcc tgt ggc ttt gac atc aac aag act ttcata 3853 Ala Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile130 135 140 ttc tct gac ctg gac tac atg ggg atg agc tca ggt ttc tac aaaaat 3901 Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn145 150 155 gtg gtg aag att caa aag cat gtt acc ttc aac caa gtg aaa ggcatt 3949 Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile160 165 170 ttc ggc ttc act gac agc gac tgc att ggg aag atc agt ttt cctgcc 3997 Phe Gly Phe Thr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala175 180 185 190 atc cag gct gct ccc tcc ttc agc aac tca ttc cca cag atcttc cga 4045 Ile Gln Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile PheArg 195 200 205 gac agg acg gat atc cag tgc ctt atc cca tgt gcc att gaccag gat 4093 Asp Arg Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp GlnAsp 210 215 220 cct tac ttt aga atg aca agg gac gtc gcc ccc agg atc ggctat cct 4141 Pro Tyr Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly TyrPro 225 230 235 aaa cca gcc ctg ttg cac tcc acc ttc ttc cca gcc ctg cagggc gcc 4189 Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln GlyAla 240 245 250 cag acc aaa atg agt gcc agc gac cca aac tcc tcc atc ttcctc acc 4237 Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe LeuThr 255 260 265 270 gac acg gcc aag cag atc aaa acc aag gtc aat aag catgcg ttt tct 4285 Asp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His AlaPhe Ser 275 280 285 gga ggg aga gac acc atc gag gag cac agg cag ttt gggggc aac tgt 4333 Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly GlyAsn Cys 290 295 300 gat gtg gac gtg tct ttc atg tac ctg acc ttc ttc ctcgag gac gac 4381 Asp Val Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu GluAsp Asp 305 310 315 gac aag ctc gag cag atc agg aag gat tac acc agc ggagcc atg ctc 4429 Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly AlaMet Leu 320 325 330 acc ggt gag ctc aag aag gca ctc ata gag gtt ctg cagccc ttg atc 4477 Thr Gly Glu Leu Lys Lys Ala Leu Ile Glu Val Leu Gln ProLeu Ile 335 340 345 350 gca gag cac cag gcc cgg cgc aag gag gtc acg gatgag ata gtg aaa 4525 Ala Glu His Gln Ala Arg Arg Lys Glu Val Thr Asp GluIle Val Lys 355 360 365 gag ttc atg act ccc cgg aag ctg tcc ttc gac tttcag aag ctt gcg 4573 Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe GlnLys Leu Ala 370 375 380 gcc gca ctc gag cac cac cac cac cac cactgagatccgg ctgctaacaa 4623 Ala Ala Leu Glu His His His His His His 385390 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct4683 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat4742 16 392 PRT Artificial Sequence Description of Artificial Sequencehuman minor TrpRS fragment in pET20B 16 Met Ser Ala Lys Gly Ile Asp TyrAsp Lys Leu Ile Val Arg Phe Gly 1 5 10 15 Ser Ser Lys Ile Asp Lys GluLeu Ile Asn Arg Ile Glu Arg Ala Thr 20 25 30 Gly Gln Arg Pro His His PheLeu Arg Arg Gly Ile Phe Phe Ser His 35 40 45 Arg Asp Met Asn Gln Val LeuAsp Ala Tyr Glu Asn Lys Lys Pro Phe 50 55 60 Tyr Leu Tyr Thr Gly Arg GlyPro Ser Ser Glu Ala Met His Val Gly 65 70 75 80 His Leu Ile Pro Phe IlePhe Thr Lys Trp Leu Gln Asp Val Phe Asn 85 90 95 Val Pro Leu Val Ile GlnMet Thr Asp Asp Glu Lys Tyr Leu Trp Lys 100 105 110 Asp Leu Thr Leu AspGln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys 115 120 125 Asp Ile Ile AlaCys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser 130 135 140 Asp Leu AspTyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val 145 150 155 160 LysIle Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly 165 170 175Phe Thr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln 180 185190 Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg 195200 205 Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr210 215 220 Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro LysPro 225 230 235 240 Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln GlyAla Gln Thr 245 250 255 Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile PheLeu Thr Asp Thr 260 265 270 Ala Lys Gln Ile Lys Thr Lys Val Asn Lys HisAla Phe Ser Gly Gly 275 280 285 Arg Asp Thr Ile Glu Glu His Arg Gln PheGly Gly Asn Cys Asp Val 290 295 300 Asp Val Ser Phe Met Tyr Leu Thr PhePhe Leu Glu Asp Asp Asp Lys 305 310 315 320 Leu Glu Gln Ile Arg Lys AspTyr Thr Ser Gly Ala Met Leu Thr Gly 325 330 335 Glu Leu Lys Lys Ala LeuIle Glu Val Leu Gln Pro Leu Ile Ala Glu 340 345 350 His Gln Ala Arg ArgLys Glu Val Thr Asp Glu Ile Val Lys Glu Phe 355 360 365 Met Thr Pro ArgLys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala 370 375 380 Leu Glu HisHis His His His His 385 390 17 6 PRT Homo sapiens 17 Glu Leu Arg Val SerTyr 1 5 18 6 PRT Escherichia coli 18 Glu Thr Val Gln Glu Trp 1 5 19 9PRT Homo sapiens 19 Ser Ala Lys Glu Leu Arg Cys Gln Cys 1 5 20 11 PRTHomo sapiens 20 Ala Ser Val Ala Thr Glu Leu Arg Cys Gln Cys 1 5 10 21 7PRT Homo sapiens 21 Ala Glu Leu Arg Cys Gln Cys 1 5 22 58 PRT Homosapiens 22 Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu Lys Lys Gly GluLys 1 5 10 15 Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser Ala Asp SerLys Pro 20 25 30 Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly Cys Ile IleThr Ala 35 40 45 Arg Lys His Pro Asp Ala Asp Ser Leu Tyr 50 55 23 58 PRTHomo sapiens 23 Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala Tyr Pro Asp ProSer Lys 1 5 10 15 Gln Lys Pro Met Ala Lys Gly Pro Ala Lys Asn Ser GluPro Glu Glu 20 25 30 Val Ile Pro Ser Arg Leu Asp Ile Arg Val Gly Lys IleIle Thr Val 35 40 45 Glu Lys His Pro Asp Ala Asp Ser Leu Tyr 50 55 24 7PRT Homo sapiens 24 Arg Val Gly Lys Ile Ile Thr 1 5 25 7 PRT Homosapiens 25 Arg Ile Gly Cys Ile Ile Thr 1 5 26 7 PRT Homo sapiens 26 ArgIle Gly Arg Ile Ile Thr 1 5 27 7 PRT Caenorhabditis elegans 27 Arg ValGly Arg Ile Ile Lys 1 5 28 7 PRT Saccharomyces cerevisiae 28 Arg Val GlyPhe Ile Gln Lys 1 5 29 7 PRT Bos taurus 29 Arg Val Gly Lys Val Ile Ser 15 30 7 PRT Mus musculus 30 Arg Ile Gly Cys Ile Val Thr 1 5 31 7 PRTMesocricetus auratus 31 Arg Ile Gly Arg Ile Val Thr 1 5 32 7 PRT Ovisaries 32 Arg Ile Gly Cys Ile Ile Thr 1 5 33 7 PRT Calcarea sp. 33 ArgIle Gly Arg Ile Thr Ser 1 5 34 7 PRT A. aeolicus 34 Arg Val Ala Lys ValLeu Ser 1 5 35 7 PRT Escherichia coli 35 Arg Val Gly Lys Ile Val Glu 1 536 7 PRT Escherichia coli 36 Arg Val Ala Leu Ile Glu Asn 1 5 37 7 PRTHaemophilus influenzae 37 Arg Val Ala Lys Val Leu Lys 1 5 38 7 PRTBacillus subtilis 38 Arg Val Ala Glu Val Ile Glu 1 5 39 7 PRT B.stearothermophilus 39 Arg Val Ala Glu Val Val Gln 1 5 40 7 PRT Thermusthermophilus 40 Arg Val Ala Glu Val Leu Ala 1 5 41 6 PRT Escherichiacoli 41 Val Gly Glu Val Val Glu 1 5 42 6 PRT Bacillus subtilis 42 IleGly His Val Leu Glu 1 5 43 6 PRT Synechococcus sp. 43 Val Gly Arg ValLeu Glu 1 5 44 6 PRT Thermus thermophilus 44 Phe Ala Arg Val Leu Glu 1 545 85 PRT Homo sapiens 45 Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp TyrLys Ala Asp Cys Pro 1 5 10 15 Pro Gly Asn Pro Ala Pro Thr Ser Asn HisGly Pro Asp Ala Thr Glu 20 25 30 Ala Glu Glu Asp Phe Val Asp Pro Trp ThrVal Gln Thr Ser Ser Ala 35 40 45 Lys Gly Ile Asp Tyr Asp Lys Leu Ile ValArg Phe Gly Ser Ser Lys 50 55 60 Ile Asp Lys Glu Leu Ile Asn Arg Ile GluArg Ala Thr Gly Gln Arg 65 70 75 80 Pro His His Phe Leu 85 46 85 PRT Bostaurus 46 Thr Ser Tyr Lys Ala Ala Thr Gly Glu Asp Tyr Lys Val Asp CysPro 1 5 10 15 Pro Gly Asp Pro Ala Pro Glu Ser Gly Glu Gly Leu Asp AlaThr Glu 20 25 30 Ala Asp Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr SerSer Ala 35 40 45 Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly SerSer Lys 50 55 60 Ile Asp Lys Glu Leu Val Asn Arg Ile Glu Arg Ala Thr GlyGln Arg 65 70 75 80 Pro His Arg Phe Leu 85 47 85 PRT Mus musculus 47 MetSer Tyr Lys Ala Ala Met Gly Glu Glu Tyr Lys Ala Gly Cys Pro 1 5 10 15Pro Gly Asn Pro Thr Ala Gly Arg Asn Cys Asp Ser Asp Ala Thr Lys 20 25 30Ala Ser Glu Asp Phe Val Asp Pro Trp Thr Val Arg Thr Ser Ser Ala 35 40 45Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Gln Pro Gly Ser Ser Lys 50 55 60Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg 65 70 7580 Pro His Arg Phe Leu 85 48 85 PRT Oryctolagus cuniculus 48 Thr Ser TyrLys Glu Ala Met Gly Glu Asp Tyr Lys Ala Asp Cys Pro 1 5 10 15 Pro GlyAsn Ser Thr Pro Asp Ser His Gly Pro Asp Glu Ala Val Asp 20 25 30 Asp LysGlu Asp Phe Val Asp Pro Trp Thr Val Arg Thr Ser Ser Ala 35 40 45 Lys GlyIle Asp Tyr Asp Lys Leu Ile Val Gln Phe Gly Ser Ser Lys 50 55 60 Ile AspLys Glu Leu Val Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg 65 70 75 80 ProHis Arg Phe Leu 85 49 86 PRT Homo sapiens 49 Ile Ser Tyr Gln Gly Arg IlePro Tyr Pro Arg Pro Gly Thr Cys Pro 1 5 10 15 Gly Gly Ala Phe Thr ProAsn Met Arg Thr Thr Lys Glu Phe Pro Asp 20 25 30 Asp Val Val Thr Phe IleArg Asn His Pro Leu Met Tyr Asn Ser Ile 35 40 45 Tyr Pro Ile His Lys ArgPro Leu Ile Val Arg Ile Gly Thr Asp Tyr 50 55 60 Lys Tyr Thr Lys Ile AlaVal Asp Arg Val Asn Ala Ala Asp Gly Arg 65 70 75 80 Tyr His Val Leu PheLeu 85 50 86 PRT Mus musculus 50 Ile Ser Tyr Gln Gly Arg Ile Pro Tyr ProArg Pro Gly Thr Cys Pro 1 5 10 15 Gly Gly Ala Phe Thr Pro Asn Met ArgThr Thr Lys Asp Phe Pro Asp 20 25 30 Asp Val Val Thr Phe Ile Arg Asn HisPro Leu Met Tyr Asn Ser Ile 35 40 45 Ser Pro Ile His Arg Arg Pro Leu IleVal Arg Ile Gly Thr Asp Tyr 50 55 60 Lys Tyr Thr Lys Ile Ala Val Asp ArgVal Asn Ala Ala Asp Gly Arg 65 70 75 80 Tyr His Val Leu Phe Leu 85 51 46PRT Homo sapiens 51 Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro ProGly Asn Pro 1 5 10 15 Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr GluAla Glu Glu Asp 20 25 30 Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser AlaLys Gly 35 40 45 52 46 PRT Bos taurus 52 Ala Ala Thr Gly Glu Asp Tyr LysVal Asp Cys Pro Pro Gly Asp Pro 1 5 10 15 Ala Pro Glu Ser Gly Glu GlyLeu Asp Ala Thr Glu Ala Asp Glu Asp 20 25 30 Phe Val Asp Pro Trp Thr ValGln Thr Ser Ser Ala Lys Gly 35 40 45 53 46 PRT Mus musculus 53 Ala AlaMet Gly Glu Glu Tyr Lys Ala Gly Cys Pro Pro Gly Asn Pro 1 5 10 15 ThrAla Gly Arg Asn Cys Asp Ser Asp Ala Thr Lys Ala Ser Glu Asp 20 25 30 PheVal Asp Pro Trp Thr Val Arg Thr Ser Ser Ala Lys Gly 35 40 45 54 46 PRTOryctolagus cuniculus 54 Glu Ala Met Gly Glu Asp Tyr Lys Ala Asp Cys ProPro Gly Asn Ser 1 5 10 15 Thr Pro Asp Ser His Gly Pro Asp Glu Ala ValAsp Asp Lys Glu Asp 20 25 30 Phe Val Asp Pro Trp Thr Val Arg Thr Ser SerAla Lys Gly 35 40 45 55 41 PRT Mus musculus 55 Ala Phe Ala Gly Glu AspPhe Lys Val Asp Ile Pro Glu Thr His Gly 1 5 10 15 Gly Glu Gly Thr GluAsp Glu Ile Asp Asp Glu Tyr Glu Gly Asp Trp 20 25 30 Ser Asn Ser Ser SerSer Thr Ser Gly 35 40 56 5 PRT Homo sapiens 56 Met Gly Asp Ala Pro 1 557 5 PRT Homo sapiens 57 Ser Asn His Gly Pro 1 5 58 5 PRT Homo sapiens58 Ser Ala Lys Gly Ile 1 5

We claim:
 1. An isolated polypeptide comprising a truncatedtryptophanyl-tRNA synthetase polypeptide comprising a Rossmann foldnucleotide binding domain, wherein the isolated polypeptide is capableof regulating vascular endothelial cell function.
 2. The isolatedpolypeptide of claim 1, wherein the truncated polypeptide has a size ofat least about 46 kilodaltons.
 3. The isolated polypeptide of claim 1,wherein the truncated tRNA synthetase polypeptide has amino-terminaltruncation.
 4. The isolated polypeptide of claim 1, wherein thepolypeptide is angiogenic.
 5. The isolated polypeptide of claim 1,wherein the polypeptide is angiostatic.
 6. The isolated polypeptide ofclaim 1, wherein the truncated tRNA synthetase polypeptide is a memberof the group consisting of a polypeptide consisting essentially of aminoacid residues 48-471 of SEQ ID NO:9; a polypeptide consistingessentially of amino acid residues 71-471 of SEQ ID NO:9; a polypeptideof approximately 47 kD molecular weight produced by cleavage of thepolypeptide of SEQ ID NO:9 with polymorphonuclear leucocyte elastase;and fragments thereof comprising the amino acid sequence -Asp-Leu-Thr-.7. The isolated polypeptide of claim 1, wherein the polypeptide ismammalian.
 8. The isolated polypeptide of claim 1, wherein thepolypeptide is human.
 9. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a polynucleotide ofSEQ ID NO:9; (b) a polynucleotide which is hybridizable to apolynucleotide of SEQ ID NO:9; (c) a polynucleotide encoding apolypeptide of claim 1; (d) a polynucleotide that is hybridizable to apolynucleotide encoding a polypeptide of claim 1; (e) a polynucleotideencoding a polypeptide of claim 6; (f) a polynucleotide that ishybridizable to a polynucleotide encoding a polypeptide of claim 6; (g)a polynucleotide encoding a polypeptide epitope of SEQ ID NO:9; and (h)a polynucleotide that is hybridizable to a polynucleotide encoding apolypeptide epitope of SEQ ID NO:9.
 10. An isolated nucleic acidmolecule of SEQ ID NO:9, wherein the nucleotide sequence comprisessequential nucleotide deletions from either the 5′-terminus or the3′-terminus.
 11. A recombinant vector comprising an isolated nucleicacid molecule of SEQ ID NO:9.
 12. A method of making a recombinant hostcell comprising introducing an isolated nucleic acid molecule of SEQ IDNO:9 into the host cell.
 13. A recombinant host cell produced by themethod of claim
 12. 14. The recombinant host cell of claim 13 comprisinga vector sequence that includes a nuclear acid molecule of SEQ ID NO:9.15. An isolated antibody that binds specifically to an isolatedpolypeptide of claim
 1. 16. A recombinant host cell that expresses anisolated polypeptide of claim
 1. 17. A method of making an isolatedpolypeptide comprising: (a) culturing the recombinant host cell of claim16 in which said polypeptide is expressed; and (b) isolating expressedpolypeptide from the cell culture.
 18. A process for producing thepolypeptide of claim 1, comprising treating tryptophanyl-tRNA synthetasewith a protease.
 19. The process of claim 18, wherein the protease ispolymorphonuclear leukocyte elastase.
 20. An isolated polypeptide whichis a truncated mammalian tryptophanyl-tRNA synthetase polypeptide havingchemokine activity.
 21. The isolated polypeptide of claim 20, whereinthe truncated polypeptide has an amino-terminal truncation.
 22. Theisolated polypeptide of claim 20, wherein the polypeptide has angiogenicactivity.
 23. The isolated polypeptide of claim 22, wherein theangiogenic activity is at least two-fold greater than control levels.24. The isolated polypeptide of claim 20, wherein the polypeptide hasangiostatic activity.
 25. The isolated polypeptide having angiostaticactivity of claim 24, wherein the polypeptide suppresses at least tenpercent of angiogenic activity.
 26. The isolated polypeptide havingangiostatic activity of claim 24, wherein the polypeptide suppresses atleast ninety percent of angiogenic activity.
 27. An isolated nucleicacid molecule that encodes the polypeptide of claim
 20. 28. Arecombinant vector comprising the isolated nucleic acid molecule ofclaim
 27. 29. A recombinant host cell comprising the isolated nucleicacid molecule of claim
 27. 30. An isolated antibody that bindsspecifically to the isolated polypeptide of claim
 20. 31. A recombinanthost cell that expresses the isolated polypeptide of claim
 20. 32. Amethod of making an isolated polypeptide comprising: (a) culturing therecombinant host cell of claim 31 in which said polypeptide isexpressed; and (b) isolating expressed polypeptide from the cellculture.
 33. A process for producing the polypeptide of claim 20,comprising treating tryptophanyl-tRNA synthetase with a protease. 34.The use of the isolated polypeptide of claim 1 for the preparation of apharmaceutical composition for transdermal, transmucosal, enteral orparenteral administration.
 35. A method of preparing a pharmaceuticalcomposition suitable for transdermal, transmucosal, enteral orparenteral administration comprising the step of combining the isolatedpolypeptide of claim 1 and a pharmaceutically suitable excipient.
 36. Acomposition comprising the isolated polypeptide of claim 1 and apharmaceutically suitable excipient.
 37. A composition comprising theisolated polypeptide of claim 6 and a pharmaceutically suitableexcipient.
 38. A composition comprising the isolated polypeptide ofclaim 20 and a pharmaceutically suitable excipient.
 39. A method ofsuppressing angiogenesis in a mammal comprising the step ofadministering to the mammal an angiostatically effective amount of thecomposition of claim
 37. 40. The method of claim 39, wherein the mammalis a human.
 41. A method of treating, in a mammal, a condition thatwould benefit from decreased angiogenesis comprising the step ofadministering to the mammal an angiostatically effective amount of thecomposition of claim
 37. 42. The method of claim 41, wherein the mammalis a human.
 43. A method of treating a solid tumor in a mammalcomprising the step of administering an angiostatically to the mammaleffective amount of the composition of claim
 37. 44. The method of claim43, wherein the mammal is a human.
 45. A method of suppressing tumormetastasis in a mammal comprising the step of administering anangiostatically effective amount of the composition of claim
 37. 46. Themethod of claim 45, wherein the mammal is a human.
 47. A method ofdiagnosing a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of claim 9; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the determined presence or absence ofsaid mutation.
 48. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 1 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thedetermined presence or amount of expression of the polypeptide.